Wnt surrogate molecules and uses thereof

ABSTRACT

The present invention provides Wnt pathway agonists and related compositions, which may be used in any of a variety of therapeutic methods for the treatment of diseases.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/607,875, filed Dec. 19, 2017, U.S. Provisional Application No.62/641,217, filed Mar. 9, 2018, and U.S. Provisional Application No.62/680,522, filed Jun. 4, 2018, each of which is incorporated byreference herein in its entirety.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is SRZN_006_03WO_ST25.txt. The text file is 1.2 MB,was created on Dec. 19, 2018, and is being submitted electronically viaEFS-Web.

BACKGROUND Technical Field

The present invention relates generally to Wnt signaling pathway agonistmolecules, compositions, and methods of using the same. Such moleculesare useful, for example, in modulating Wnt signaling pathways.

Description of the Related Art

Wnt (“Wingless-related integration site” or “Wingless and Int-1” or“Wingless-Int”) ligands and their signals play key roles in the controlof development, homeostasis and regeneration of many essential organsand tissues, including bone, liver, skin, stomach, intestine, kidney,central nervous system, mammary gland, taste bud, ovary, cochlea andmany other tissues (reviewed, e.g., by Clevers, Loh, and Nusse, 2014;346:1248012). Modulation of Wnt signaling pathways has potential fortreatment of degenerative diseases and tissue injuries.

One of the challenges for modulating Wnt signaling as a therapeutic isthe existence of multiple Wnt ligands and Wnt receptors, Frizzled 1-10(Fzd1-10), with many tissues expressing multiple and overlapping Fzds.Canonical Wnt signals also involve Low-density lipoprotein (LDL)receptor-related protein 5 (LRP5) or Low-density lipoprotein (LDL)receptor-related protein 6 (LRP6) as co-receptors, which are broadlyexpressed in various tissues, in addition to Fzds. Accordingly, there isclearly a need in the art for binding moieties that specifically bind toone or more Fzd, LRP5, or LRP6 to modulate Wnt signaling pathways. Thepresent invention addresses this need.

BRIEF SUMMARY

In various embodiments, the present invention provides WNT surrogatemolecules and related uses thereof.

In one embodiment, the disclosure provides a soluble, bivalent,bispecific Wnt surrogate molecule, wherein the Wnt surrogate moleculecomprises: (i) one or more regions that specifically binds to one ormore Frizzled (Fzd) receptor (a Fzd binding region); and (ii) one ormore regions that specifically binds to a Low-density lipoprotein (LDL)receptor-related protein 5 (LRP5) and/or a Low-density lipoprotein (LDL)receptor-related protein 6 (LRP6) (a LRP5/6 binding region).

In particular embodiments, the Wnt surrogate molecule comprises two ormore Fzd binding regions and two or more LRP5/6 binding regions. Inparticular embodiments, one or more Fzd binding regions comprise one ormore antigen-binding fragments of an antibody. In particularembodiments, one or more antigen-binding fragments are selected from thegroup consisting of: IgG, scFv, Fab, and VHH or sdAb

In particular embodiments, any of the Fzd antigen-binding fragmentscomprise: (i) CDRH1, CDRH2 and CDRH3 sequences set forth for any of theantibodies of Tables 1A or 1B; and/or (ii) CDRL1, CDRL2 and CDRL3sequences set forth for any of the antibodies of Tables 1A or 1B, or avariant of said Fzd binding region comprising one or more amino acidmodifications, wherein said variant comprises less than 8 amino acidsubstitutions in said CDR sequences. In particular embodiments, any ofthe Fzd binding regions comprise an amino acid sequence having at least90% identity to any of the sequences set forth in SEQ ID NOs:1-65 or129-132, or an antigen-binding fragment thereof.

In particular embodiments, any of the Fzd binding regions bind to one ormore of Frizzled 1 (Fzd1), Frizzled 2 (Fzd2), Frizzled 3 (Fzd3),Frizzled 4 (Fzd4), Frizzled 5 (Fzd5), Frizzled 6 (Fzd6), Frizzled 7(Fzd7), Frizzle 8 (Fzd8), Frizzled 9 (Fzd9), and Frizzled 10 (Fzd10). Inparticular embodiments, any of the Fzd binding region binds to two ormore of Frizzled 1 (Fzd1), Frizzled 2 (Fzd2), Frizzled 3 (Fzd3),Frizzled 4 (Fzd4), Frizzled 5 (Fzd5), Frizzled 6 (Fzd6), Frizzled 7(Fzd7), Frizzled 8 (Fzd8), Frizzled 9 (Fzd9), and Frizzled 10 (Fzd10).In particular embodiments, any of the Fzd binding region binds to: (i)Fzd1, Fzd2, Fzd7 and Fzd9; (ii) Fzd1, Fzd2 and Fzd7; (iii) Fzd5 andFzd8; (iv) Fzd5, Fzd7 and Fzd8; (v) Fzd1, Fzd4, Fzd5 and Fzd8; (vi)Fzd1, Fzd2, Fzd5, Fzd7 and Fzd8; (vii) Fzd4 and Fzd9; (viii) Fzd9 andFzd10; (ix) Fzd5, Fzd8 and Fzd10; or (x) Fzd4, Fzd5 and Fzd8; Fzd1,Fzd5, Fzd7 and Fzd8.

In particular embodiments, any of the surrogate molecules comprise oneor more LRP5/6 binding regions comprise one or more antigen-bindingfragments of an antibody. In particular embodiments, the one or moreantigen-binding fragments are selected from the group consisting of:IgG, scFv, Fab, and VHH or sdAb In particular embodiments, any of theone or more LRP5/6 binding regions or antigen-binding fragmentscomprise: (i) CDRH1, CDRH2 and CDRH3 sequences set forth for any of theantibodies of Table 2; and/or (ii) CDRL1, CDRL2 and CDRL3 sequences setforth for any of the antibodies of Table 2, or a variant of said LRP5/6binding region comprising one or more amino acid modifications, whereinsaid variant comprises less than 8 amino acid substitutions in said CDRsequences. In particular embodiments, any of the one or more LRP5/6binding regions comprise an amino acid sequence having at least 90%identity to any of the sequences set forth in SEQ ID NOs:66-88 or 133,or an antigen-binding fragment thereof.

In particular embodiments of any of the Wnt surrogate molecules, the Fzdbinding region comprising a Fab, and the LRP5/6 binding regioncomprising a VHH or sdAb In particular embodiments, the Fab is presentwithin a full immunoglobulin (Ig), optionally an IgG, comprising a lightchain and a heavy chain. In particular embodiments, the LRP5/6 bindingregion is fused to the N-terminus or the C-terminus of the heavy chain.In particular embodiments, the LRP5/6 binding region is fused to theN-terminus or the C-terminus of the light chain. In certain embodiments,the LRP5/6 binding region is fused to the N-terminus of the heavy chainof the full Ig or the N-terminus of the light chain of the full Ig. Incertain embodiments, the LRP5/6 binding region is fused to theC-terminus of the heavy chain of the full Ig or the C-terminus of thelight chain of the full Ig. In certain embodiments, the variable lightchain region of the LRP5/6 binding Fab is fused to the N-terminus of thevariable heavy chain region of the full Ig. In certain embodiments, thevariable light chain region of the LRP5/6 binding Fab is fused to theN-terminus of the variable heavy chain region of the full Ig, and thevariable heavy chain region of the LRP5/6 binding Fab is fused to theN-terminus of the variable light chain region of the full IgG. Inparticular embodiments, any of the LRP5/6 binding region is fused to theheavy chain or the light chain via one or more linker moiety.

In certain embodiments of any of the Wnt surrogate molecules, the Fzdbinding region comprises a VHH or sdAb and the LRP5/6 binding regioncomprises a Fab. In particular embodiments, the Fab is present within afull immunoglobulin (Ig), optionally an IgG, comprising a light chainand a heavy chain. In certain embodiments, the Fzd binding region isfused to the N-terminus or the C-terminus of the heavy chain. In certainembodiments, the Fzd binding region is fused to the N-terminus or theC-terminus of the light chain. In some embodiment, the Fzd bindingregion is fused to the N-terminus of the heavy chain of the full Ig orthe N-terminus of the light chain of the full Ig. In some embodiments,the Fzd binding region is fused to the C-terminus of the heavy chain ofthe full Ig or the C-terminus of the light chain of the full Ig. In someembodiments, the variable light chain region of the Fzd binding Fab isfused to the N-terminus of the variable heavy chain region of the fullIg. In some embodiments, the variable light chain region of the Fzdbinding Fab is fused to the N-terminus of the variable heavy chainregion of the full Ig, and the variable heavy chain region of the Fzdbinding Fab is fused to the N-terminus of the variable light chainregion of the full IgG. In certain embodiments, any of the Fzd bindingregion is fused to the heavy chain or the light chain via one or morelinker moiety.

In another embodiment, any of the Fzd binding region comprises a Fab orFv, and the LRP5/6 binding region comprises a Fab or Fv. In particularembodiments, the Fab of the Fzd binding region or the Fab or Fv of theLRP5/6 binding region is present within a full immunoglobulin (Ig),optionally an IgG, comprising a light chain and a heavy chain. Inparticular embodiments, only one of the Fab of the Fzd binding region orthe Fab of the LRPp5/6 binding region is present within the fullimmunoglobulin (Ig). In particular embodiments, the Fab or Fv of the Fzdbinding region is present within the full Ig. In particular embodiments,the Fab or Fv of the LRP5/6 binding region is fused to the N-terminus ofthe Ig. In particular embodiments, the Fab of the LRP5/6 binding regionis fused to the C-terminus of the Ig. In a further embodiment, the Fabof the LRP5/6 binding region is present within the full Ig. Inparticular embodiments, the Fab of the Fzd binding region is fused tothe N-terminus of the Ig. In particular embodiments, the Fab of the Fzdbinding region is fused to the C-terminus of the Ig. In someembodiments, the variable light chain region of the Fzd binding Fab isfused to the N-terminus of the variable heavy chain region of the fullIg, and the variable heavy chain region of the Fzd binding Fab is fusedto the N-terminus of the variable light chain region of the full IgG. Insome embodiments, the variable light chain region of the LRP5/6 bindingFv is fused to the N-terminus of the variable heavy chain region of thefull Ig, and the variable heavy chain region of the LRP5/6 binding Fv isfused to the N-terminus of the variable light chain region of the fullIgG. In some embodiments, the variable heavy chain region of the LRP5/6binding Fv is fused to the N-terminus of the variable heavy chain regionof the full Ig, and the variable light chain region of the LRP5/6binding Fv is fused to the N-terminus of the variable light chain regionof the full IgG. In some embodiments, variable light chain region of theFzd binding Fv is fused to the N-terminus of the variable heavy chainregion of the full Ig, and the variable heavy chain region of the Fzdbinding Fv is fused to the N-terminus of the variable light chain regionof the full IgG. In some embodiments, the variable heavy chain region ofthe Fzd binding Fv is fused to the N-terminus of the variable heavychain region of the full Ig, and the variable light chain region of theFzd binding Fv is fused to the N-terminus of the variable light chainregion of the full IgG.

In another embodiment, any of the Fzd binding regions comprises a VHH orsdAb, and the LRP5/6 binding region comprises a VHH or sdAb. Inparticular embodiments, the Fzd binding region is fused to the Lrp5/6binding region, and wherein the Fzd binding region or the LRP5/6 bindingregion is fused to an Fc region. In particular embodiments, the Fzdbinding region is fused to the N-terminus of an Fc region, and whereinthe LRP5/6 binding region is fused to the C-terminus of an Fc region. Inparticular embodiments, the Fzd binding region is fused to theC-terminus of an Fc region, and wherein the LRP5/6 binding region isfused to the N-terminus of an Fc region.

In a further embodiment, any of the antibodies or one or moreantigen-binding fragment thereof, is humanized. In a further embodiment,any of the Wnt surrogate molecules binds to one or more Fzd receptorwith a K_(D) of 50 μM or lower. In further embodiments, any of the Wntsurrogate molecules binds to LRP5 and/or LRP6 with a K_(D) of 50 μM orlower.

In a further embodiment, any of the Wnt surrogate molecule modulates aWnt signaling pathway in a cell, optionally a mammalian cell. Inparticular embodiments, the Wnt surrogate molecule increases signalingvia the Wnt signaling pathway in the cell. In particular embodiments,the Wnt signaling pathway is a canonical Wnt signaling pathway or anon-canonical Wnt signaling pathway.

In a related embodiment, the present disclosure provides an isolatedpolynucleotide encoding a polypeptide sequence comprising one or more ofthe Fzd binding regions and/or one or more of the LRP5/6 binding regionsof a Wnt surrogate molecule. In particular embodiments, the presentdisclosure provides an expression vector comprising the isolatedpolynucleotide. In further particular embodiments, the presentdisclosure provides an isolated host cell comprising the expressionvector.

In a related embodiment, the present disclosure provides apharmaceutical composition comprising a physiologically acceptableexcipient, diluent, or carrier, and a therapeutically effective amountof any of the Wnt surrogate molecules disclosed herein.

In related embodiments, the present disclosure provides a method foragonizing a Wnt signaling pathway in a cell, comprising contacting thecell any of the Wnt surrogate molecules, wherein the Wnt surrogatemolecule is an agonist of a Wnt signaling pathway.

In particular embodiments, the present disclosure provides a method fortreating a subject having a disease or disorder associated with reducedWnt signaling, comprising administering to the subject an effectiveamount of the pharmaceutical composition, wherein the Wnt surrogatemolecule is an agonist of a Wnt signaling pathway. In particularembodiments, the disease or disorder is selected from the groupconsisting of: bone fractures, osteoporosis (e.g., post-menopausalosteoporosis), osteoporotic fractures, spinal fusion, vertebralcompression fracture, pre-operative spinal surgery optimization,osseointegration of orthopedic devices, tendon-bone integration, toothgrowth and regeneration, dental implantation, periodontal diseases,maxillofacial reconstruction, osteonecrosis of the jaw, osteoarthritis(OA), muscular dystrophy, muscle atrophy resulting from sarcopenia orcachexia, alopecia, hearing loss, including regeneration of inner andouter auditory hair cells, vestibular hypofunction, maculardegeneration, vitreoretinopathy, diseases of retinal degeneration,including diabetic retinopathy, diseases/disorders affecting theintegrity of the blood brain barrier, Fuchs' dystrophy, stroke,traumatic brain injury, Alzheimer's disease, multiple sclerosis, spinalcord injuries, oral mucositis, short bowel syndrome, inflammatory boweldiseases (IBD), including Crohn's disease (CD) and ulcerative colitis(UC), in particular IBD with fistula formation, metabolic syndrome,diabetes, dyslipidemia, pancreatitis, exocrine pancreatic insufficiency,wound healing, diabetic foot ulcers, coronary artery disease, acutekidney injuries, chronic kidney diseases, chronic obstructive pulmonarydiseases (COPD), pulmonary fibrosis, including idiopathic pulmonaryfibrosis, acute liver failure, acute alcoholic liver injuries, chronicliver diseases with hepatitis C virus (HCV), HCV subjects post-antiviraldrug therapies, chronic liver diseases with hepatitis B virus (HBV),fibrosis, HBV subjects post-antiviral drug therapies, chronic alcoholicliver diseases, alcoholic hepatitis, non-alcoholic fatty liver diseasesand non-alcoholic steatohepatitis (NASH), cirrhosis, and chronic liverinsufficiencies of all causes. In certain embodiments, the disease ordisorder is a bone disease or disorder. In particular embodiments, thedisease or disorder is a bone disease or disorder, and the Wnt surrogatemolecule binds Fzd1, Fzd2, and FZD7, and binds LRP5 and/or LRP6. Incertain embodiments, the disease or disorder is a bone disease ordisorder, and the Wnt surrogate molecule binds Fzd1, Fzd2, FZD7, Fzd5and Fzd8, and also binds LRP5 and/or LRP6.

In another related embodiment, the present disclosure provides a methodfor increasing bone mineral density, increasing bone volume, increasingbone cortical thickness, increasing bone mineral apposition rate,increasing bone stiffness, increasing bone biomechanical strength,increasing resistance to bone fracture, decreasing bone resorption, ordecreasing bone loss associated with osteoporosis, in a subject in needthereof, comprising providing to the subject an effective amount of apharmaceutical composition comprising a Wnt surrogate molecule, whereinthe Wnt surrogate molecule is an agonist of a Wnt signaling pathway. Incertain embodiments, the Wnt surrogate molecule binds Fzd1, Fzd2, andFZD7, and binds LRP5 and/or LRP6. In certain embodiments, the Wntsurrogate molecule binds Fzd1, Fzd2, FZD7, Fzd5 and Fzd8, and also bindsLRP5 and/or LRP6.

In particular embodiments, methods of the invention, including thoserelated to treating or preventing a bone disease or disorder, such asosteoporosis (e.g., post-menopausal osteoporosis), further compriseproviding the subject an antiresorptive agent (in combination with a Wntsurrogate molecule). Examples of anti-resorptive agents include, but arenot limited to, bisphosphonates or selective estrogen receptormodulators. Antiresorptive agents are used to increase bone strength inindividuals with osteoporosis and include five principal classes ofagents: bisphosphonates, estrogens, selective estrogen receptormodulators (SERMs), calcitonin and monoclonal antibodies such asdenosumab, any of which may be used. Illustrative examples ofantiresorptive agents include, but are not limited to: bisphosphonates,e.g., alendronate-generic medication (Brand name: Fosamax™, Fosamax™Plus D), risedronate (Brand name: Actonel™, Actonel™ with Calcium),ibandronate (Brand name: Boniva™), and zoledronic acid (Brand name:Reclast™); other antiresorptives, e.g., estrogen therapy or hormonetherapy, raloxifene (Brand name: Evista™), and denosumab (Prolial™); andanabolic medication, e.g., teriparatide (Forteo™).

In a further related embodiment, the present disclosure provides amethod for increasing liver to body weight ratio, promoting liverregeneration, increasing liver cell proliferation or mitosis, decreasingliver fibrosis, optionally following a chronic liver injury, increasinghepatocyte function, or decreasing coagulation time in liver, in asubject in need thereof, comprising providing to the subject aneffective amount of a pharmaceutical composition comprising a Wntsurrogate molecule, wherein the Wnt surrogate molecule is an agonist ofa Wnt signaling pathway.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-D. Schematic diagrams of illustrative formats of Wnt surrogatemolecules.

FIGS. 2A-2D. Characterization of a Wnt surrogate molecule, R2M3-26.

FIGS. 3A-3D. Characterization of a Wnt surrogate molecule, R2M3-32.

FIGS. 4A-4B. Graphs showing that R2M3-26 and R2M3-32 activities can beinhibited by soluble Fzd ECD and by R2M3 IgG alone without the Lrpbinding arm.

FIG. 5. Characterization of illustrative R2M3-Lrp6 binder fusions in293, Huh7, A375, and BNL.CL2 Wnt dependent reporter assays.

FIG. 6. Characterization of illustrative 18R5-Lrp6 binder fusions in293, A375, and BNL.CL2 Wnt dependent reporter assays.

FIG. 7. Characterization of illustrative 18R5-Lrp5 binder fusions in 293Wnt dependent reporter assays.

FIG. 8A-8B. Characterization of illustrative Fzd binders-Lrp6 binder 26fusions in 293 Wnt dependent reporter assays.

FIG. 9. SAR analysis of illustrative Wnt surrogate molecules in theIgG-Nab fusion format.

FIG. 10A-10B. Characterization of R2M3-26 in the Fab format in 293 Wntdependent reporter assays.

FIG. 11A-11B. Characterization of R2M3-32 in the Fab format in 293 Wntdependent reporter assays.

FIG. 12A-12B. Characterization of R2M3-26 in the Hetero-Ig format in 293Wnt dependent reporter assays.

FIG. 13. Characterization of 26-17SB9 in the VHH/sdAb-VHH/sdAb format,in different tandem formats, and on different ends of the Fc fragment in293 Wnt dependent reporter assays.

FIG. 14A-14H. Characterization of 18R5-LRP6 Binder Fusions in tandemscFv formats in 293 Wnt dependent reporter assays.

FIG. 15A-15G. Characterization of various Wnt Surrogate molecules in theFab-IgG format in 293 Wnt dependent reporter assays.

FIG. 16A-16C. Characterization of R2M3-26 in the F(ab′)2 format in 293Wnt dependent reporter assays.

FIG. 17A-17H. Characterization of additional Wnt surrogate molecules in293 Wnt dependent reporter assays

FIG. 18A-18C. A. Schematic diagram of the 2Fv-Ig format. B-C.Characterization of the Wnt surrogate molecule, 10SG11-1RC07.

FIG. 19. Sequences of polypeptide chains of illustrative Wnt surrogatesmolecules.

FIG. 20A-20B. In vivo PK/PD characterization of R2M3-26

FIGS. 21A-21E. Images and graphs showing that systemic expression of18R5-DKK1c for 14 days results in increased bone mineral density. *Pvalue <0.05; ** P value <0.0001. For each time point, the bars from leftto right are as follows: vehicle (diamond), romosozumab (square), AAVCAG-GFP (triangle), AAV ScFv (anti-GFP)-DKK1cF234K-Flag-His (invertedtriangle), and AAV 18R5-DKK1c-FLagHis (circle).

FIGS. 22A-22D. Images and graphs showing that systemic expression of18R5-DKK1c for 14 days or 28 days results in increased bone volume. Foreach time point, the bars from left to right are as follows: vehicle,romosozumab, AAV CAG-GFP, AAV ScFv (anti-GFP)-DKK1cF234K-Flag-His, andAAV 18R5-DKK1c-FLagHis. *P value <0.05; ** P value <0.0001, ****P value<0.0001.

FIGS. 23A-23B. Graphs showing the dynamic parameters of bone formationbased on fluorochrome labelling. For each time point, the bars from leftto right are as follows: vehicle, romosozumab, AAV CAG-GFP, AAV ScFv(anti-GFP)-DKK1cF234K-Flag-His, and AAV 18R5-DKK1c-FlagHis.

FIGS. 24A-24D. Graphs and images showing that systemic expression of18R5-DKK1c results in increased osteoblast and reduced osteoclast onbone surface. For each time point, the bars from left to right are asfollows: vehicle, romosozumab, AAV CAG-GFP, AAV ScFv(anti-GFP)-DKK1cF234K-Flag-His, and AAV 18R5-DKK1c-FlagHis. ** P value<0.05.

FIGS. 25A-25C. Diagram of assay for bone stiffness and fracture andgraphs showing ultimate load to failure and stiffness in mice treated asindicated.

FIGS. 26A-26D. Graphs and images showing that systemic treatment withR2M3-26 results in rapid and sustained increase in bone after one week.For each timepoint, the bars from left to right correspond to thetreatments indicated from top to bottom. **** indicates P value <0.0001.

FIGS. 27A-27C. Images and graphs showing that R2M3-26 treatment rapidlyreverses the bone loss associated with ovariectomy-induced osteoporosis.For each time point, the bars from left to right correspond to thetreatments indicated from top to bottom.

FIGS. 28A-28C. Images and graphs showing that a single injection ofR2M3-26 rapidly increases bone volume.

FIGS. 29A-29D. Graphs showing that high doses of R2M3-26 and 1R-C07-26significantly and rapidly increase bone formation in naïve mice. Foreach timepoint, the bars from left to right correspond to the treatmentsindicated from top to bottom.

FIG. 30. Graphs showing that R2M3-26 and 1R-C07-3 increase bone mineraldensity in naïve mice. For each timepoint, the bars from left to rightcorrespond to the treatments indicated from top to bottom.

FIG. 31. Graph showing changes in whole body bone mineral density (BMD)measured weekly in ovariectomized mice as compared with naïve and shamsurgery operated mice.

FIG. 32. Changes in vertebral mineral density (image shown) and changesthe vertebral resistance to compression fracture in vertebra isolatedfrom mice after various treatments (bar graph) as measured in newtonunits of force (N) after 4 weeks of treatment.

FIGS. 33A-D. Test of Wnt surrogate molecules in an Einhorn fracturemodel. Radiographs of the callus after 1 week (A) and 6 weeks (B) oftreatment with Wnt surrogate molecules are shown. Graphs of changes inwhole body bone mineral density (BMD) in contralateral femur are shown(C). Scatter plots showing changes in callus tissue volume, callus bonevolume, bone volume/tissue volume ratio (BV/TV), and bone mineralcontent per millimeter (BMC/mm) as shown in (D) along withrepresentative images of bone slices are shown.

FIG. 34. Graph with changes in whole body bone mineral density (BMD)measured weekly with different Wnt surrogate molecule dosing schedulesis shown.

FIG. 35. Graph with changes in whole body bone mineral density (BMD)measured weekly from mice treated with different Wnt surrogate moleculealone and in combination with Romosozumab is shown.

FIG. 36. Levels of therapeutic molecules in serum as measured by ELISA.These data accompany gene expression data presented in Table 4.

FIGS. 37A-37C. Liver (A), small intestine (B) and colon (C) to bodyweight ratio after treatment with AAV-delivered Wnt surrogates. (**)p<0.01. For each graph, the treatments shown from left to rightcorrespond to those in the legend from top to bottom.

FIGS. 38A-38B. Body weight (A) and liver to body weight (B) ratio aftertreatment with recombinantly produced Wnt surrogate proteins. (*)p<0.05. For each time point, the treatments shown from left to rightcorrespond to those in the legend from top to bottom.

FIGS. 39A-39D. Induction of proliferation markers in response to R2M3-26and Rspo2 recombinant proteins. Liver Ki67 (A) and cyclinD1 (B) mRNAexpression. Average count of PCNA (C) or phospho-histone H3 (D) positivenuclei per 10× field after immunohistochemistry staining with PCNA andphospho-histone H3 antibodies respectively. (*) p<0.05, (**) p<0.01,(***) p<0.001, (****) p<0.0001. For each time point, the treatmentsshown from left to right correspond to those in the legend from top tobottom.

FIGS. 40A-40H. Efficacy of AAV-delivered Wnt surrogate and R-Spondin ina thioacetamide-induced chronic liver disease model. Design for study 1(A) and study 2 (B). Liver to body weight ratio (C-D), liver weight(E-F), liver collagen A1 mRNA expression (G) and percentage red area inliver histological sections stained with Sirius red (H) in response toAAV-delivered wnt surrogate and R-spondin in study 1 (C, E, G and H) andstudy 2 (D, F, H). (*) p<0.05, (**) p<0.01, (***) p<0.001, (****)p<0.0001. For each graph, the treatments shown from left to rightcorrespond to those in the legend from top to bottom (not includingbaseline).

FIGS. 41A-41N. Efficacy of recombinantly produced Wnt surrogate andR-Spondin in a thioacetamide-induced chronic liver disease model. Studydesign (A). D-2, D0, D3, D7, D10, D14 represents days relative to thestart of treatment with recombinant proteins. Liver to body weight ratio(B, C), liver axin2 mRNA (D, E), cyclinD1 mRNA (F, G) and Ki67 mRNA (H,I) expression, average count of PCNA (J, K) or phospho-histone H3 (L, M)positive nuclei per 10× field after immunohistochemistry staining withPCNA and phospho-histone H3 antibodies respectively, in a study usingRspo2 mono treatment (B, D, F, H, J, L) or R2M3-26/Rspo2 combinationtreatment (C, E, G, I, K, M). Pro-thrombin time ratio relative to theaverage pro-thrombin time in plasma collected from control naïve micewithout TAA exposure (N). (*) p<0.05, (**) p<0.01, (****) p<0.0001. NoTAA treatment is indicated by the dashed line. For each bar graphtimepoint, the treatments shown from left to right correspond to thosein the legend shown above from top to bottom.

FIGS. 42A-42C. Efficacy of recombinantly produced Wnt surrogate andR-Spondin in a CC14-induced chronic liver disease model. Study design(A). Liver to body weight ratio (B) pro-thrombin time (C) and sirius redstaining (D) in response to CC14 treatment, R2M3-26 and Rspo2. (*)p<0.05, (**) p<0.01, (***) p<0.001, (****) p<0.0001. For each graph, thetreatments shown from left to right correspond to those in the legendfrom top to bottom (not including baseline).

FIGS. 43A-43D. Induction of proliferation markers in response torecombinantly produced Wnt surrogate in an acetaminophen-induced acuteliver injury model. Study design (A). Serum level of alanine transferaseat 24 and 48 hours after treatment with acetaminophen (B). RelativecyclinD1 (C) and Ki67 (D) mRNA expression. (*) p<0.05, (***) p<0.001,(****) p<0.0001.

FIGS. 44A-44D. Induction of proliferation markers in response toR-Spondin in an acetaminophen-induced acute liver injury model. Studydesign (A). Serum level of alanine transferase at 24 and 48 hours aftertreatment with acetaminophen (B). Relative cyclinD1 (C) and Ki67 (D)mRNA expression. (**) p<0.01, (***) p<0.001, (****) p<0.0001.

FIGS. 45A-45D. Induction of proliferation markers in response to Wntsurrogate and R-Spondin in an acetaminophen-induced acute liver injurymodel. Study design (A). Serum level of alanine transferase at 24, 36,48 and 60 hours after treatment with acetaminophen (B). RelativecyclinD1 (C) and Ki67 (D) mRNA expression. (*) p<0.05, (****) p<0.0001.For each time point, the treatments shown from left to right correspondto those in the legend from top to bottom.

FIGS. 46A-46D. Efficacy of recombinantly produced Wnt surrogate andR-Spondin on the survival of mice after acetaminophen-induced liverinjury. Study design (A). Survival curve of mice treated with thecontrol anti-eGFP control protein or R2M3-26 (B), Rspo2 (C) or acombination of R2M3-26 and Rspo2 (D) recombinant proteins.

DETAILED DESCRIPTION

The present disclosure relates to Wnt surrogate molecules that bind toone or more Fzd receptor and one or more LRP5 or LRP6 receptor andmodulate a downstream Wnt signaling pathway. In particular embodiments,the Wnt surrogate molecules activate a Wnt signaling pathway or increasesignaling via a Wnt signaling pathway. In particular embodiments, theWnt surrogate molecules disclosed herein comprise: (i) one or moreantibodies or antigen-binding fragments thereof that specifically bindto one or more Fzd receptor, including antibodies or antigen-bindingfragments thereof having particular Fzd receptor specificity and/orfunctional properties; and (ii) one or more antibodies orantigen-binding fragments thereof that specifically bind to LRP5 and/orLRP6. Certain embodiments encompass specific structural formats orarrangements of the Fzd binding region(s) and LRP5/6 binding region(s)of Wnt surrogate molecules advantageous in increasing downstream Wntpathway signaling and related biological effects.

Embodiments of the invention pertain to the use of Wnt surrogatemolecules for the diagnosis, assessment and treatment of diseases anddisorders associated with Wnt signaling pathways. In certainembodiments, the subject Wnt surrogate molecules are used to modulate aWnt signaling pathway in a cell or tissue. In certain embodiments, thesubject Wnt surrogate molecules are used in the treatment or preventionof diseases and disorders associated with aberrant or deregulated (e.g.,reduced) Wnt signaling, or for which modulating, e.g., increasing, Wntsignaling would provide a therapeutic benefit.

The practice of the present invention will employ, unless indicatedspecifically to the contrary, conventional methods of virology,immunology, microbiology, molecular biology and recombinant DNAtechniques within the skill of the art, many of which are describedbelow for the purpose of illustration. Such techniques are explainedfully in the literature. See, e.g., Current Protocols in MolecularBiology or Current Protocols in Immunology, John Wiley & Sons, New York,N.Y. (2009); Ausubel et al., Short Protocols in Molecular Biology, 3rded., Wiley & Sons, 1995; Sambrook and Russell, Molecular Cloning: ALaboratory Manual (3rd Edition, 2001); Maniatis et al. MolecularCloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach,vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed.,1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985);Transcription and Translation (B. Hames & S. Higgins, eds., 1984);Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guideto Molecular Cloning (1984) and other like references.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise.

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

Each embodiment in this specification is to be applied mutatis mutandisto every other embodiment unless expressly stated otherwise.

Standard techniques may be used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques may beperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. These and relatedtechniques and procedures may be generally performed according toconventional methods well known in the art and as described in variousgeneral and more specific references that are cited and discussedthroughout the present specification. Unless specific definitions areprovided, the nomenclature utilized in connection with, and thelaboratory procedures and techniques of, molecular biology, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well-known and commonly used in theart. Standard techniques may be used for recombinant technology,molecular biological, microbiological, chemical syntheses, chemicalanalyses, pharmaceutical preparation, formulation, and delivery, andtreatment of subjects.

Embodiments of the present invention relate to antibodies andantigen-binding fragments thereof that bind to one or more Fzd receptor.Sequences of illustrative antibodies, or antigen-binding fragments, orcomplementarity determining regions (CDRs) thereof, are set forth in SEQID NOs:1-65 or 129-132, Tables 1A and 1B, and Table 3. Anti-Fzdantibodies and antigen-binding fragments there that may be used orpresent in the Wnt surrogate molecules disclosed herein include, but arenot limited to, those described in the U.S. provisional application No.62/607,877, titled Anti-Frizzled Antibodies and Methods of Use, Attorneydocket no. SRZN-004/000S, filed on Dec. 19, 2017.

Embodiments of the present invention relate to antibodies andantigen-binding fragments thereof that bind to LRP5 and/or LRP6.Sequences of illustrative antibodies, or antigen-binding fragments, orcomplementarity determining regions (CDRs) thereof, are set forth in SEQID NOs:66-88 or 133, Tables 2A and 2B, and Table 3. Anti-LRP5/6antibodies and antigen-binding fragments there that may be used orpresent in the Wnt surrogate molecules disclosed herein include, but arenot limited to, those described in the U.S. provisional application No.62/607,879, titled Anti-LRP5/6 Antibodies and Methods of Use, Attorneydocket no. SRZN-005/000S, filed on Dec. 19, 2017.

As is well known in the art, an antibody is an immunoglobulin moleculecapable of specific binding to a target, such as a carbohydrate,polynucleotide, lipid, polypeptide, etc., through at least one epitoperecognition site, located in the variable region of the immunoglobulinmolecule. As used herein, the term encompasses not only intactpolyclonal or monoclonal antibodies, but also fragments thereof (such asdAb, Fab, Fab′, F(ab′)2, Fv), single chain (scFv), VHH or sdAb,synthetic variants thereof, naturally occurring variants, fusionproteins comprising an antibody or an antigen-binding fragment thereof,humanized antibodies, chimeric antibodies, and any other modifiedconfiguration of the immunoglobulin molecule that comprises anantigen-binding site or fragment (epitope recognition site) of therequired specificity. “Diabodies” or 2scFV-Ig antibodies, aremultivalent or multispecific fragments constructed by gene fusion(WO94/13804; P. Holliger et al., Proc. Natl. Acad. Sci. USA 906444-6448, 1993) are also a particular form of antibody contemplatedherein. Minibodies comprising a scFv joined to a CH3 domain are alsoincluded herein (S. Hu et al., Cancer Res., 56, 3055-3061, 1996). Seee.g., Ward, E. S. et al., Nature 341, 544-546 (1989); Bird et al.,Science, 242, 423-426, 1988; Huston et al., PNAS USA, 85, 5879-5883,1988); PCT/US92/09965; WO94/13804; P. Holliger et al., Proc. Natl. Acad.Sci. USA 90 6444-6448, 1993; Y. Reiter et al., Nature Biotech, 14,1239-1245, 1996; S. Hu et al., Cancer Res., 56, 3055-3061, 1996.

The term “antigen-binding fragment” as used herein refers to apolypeptide fragment that contains at least one CDR of an immunoglobulinheavy and/or light chain, or of a VHH or sdAb, that binds to the antigenof interest, in particular to one or more Fzd receptor or LRP5 or LRP6receptor. In this regard, an antigen-binding fragment of the hereindescribed antibodies may comprise 1, 2, 3, 4, 5, or all 6 CDRs of a VHand VL sequence set forth herein from antibodies that bind one or moreFzd receptor or LRP5 and/or LRP6. In particular embodiments, anantigen-binding fragment may comprise all three VH CDRs or all three VLCDRs. Similarly, an antigen binding fragment thereof may comprise allthree CDRs of a VHH or sdAb. An antigen-binding fragment of aFzd-specific antibody is capable of binding to a Fzd receptor. Anantigen-binding fragment of a LRP5/6-specific antibody is capable ofbinding to a LRP5 and/or LRP6 receptor. As used herein, the termencompasses not only isolated fragments but also polypeptides comprisingan antigen-binding fragment of an antibody disclosed herein, such as,for example, fusion proteins comprising an antigen-binding fragment ofan antibody disclosed herein, such as, e.g., a fusion protein comprisinga VHH or sdAb that binds one or more Fzd receptors and a VHH or sdAbthat binds LRP5 and/or LRP6.

The term “antigen” refers to a molecule or a portion of a moleculecapable of being bound by a selective binding agent, such as anantibody, and additionally capable of being used in an animal to produceantibodies capable of binding to an epitope of that antigen. In certainembodiments, a binding agent (e.g., a Wnt surrogate molecule or bindingregion thereof) is said to specifically bind an antigen when itpreferentially recognizes its target antigen in a complex mixture ofproteins and/or macromolecules. In certain embodiments, a Wnt surrogatemolecule or binding region thereof (e.g., an antibody or antigen-bindingfragment thereof) is said to specifically bind an antigen when theequilibrium dissociation constant is ≤10⁻⁷ or ≤10⁻⁸ M. In someembodiments, the equilibrium dissociation constant may be ≤10⁻⁹ M or≤10⁻¹⁰ M.

In certain embodiments, antibodies and antigen-binding fragments thereofas described herein include a heavy chain and a light chain CDR set,respectively interposed between a heavy chain and a light chainframework region (FR) set which provide support to the CDRs and definethe spatial relationship of the CDRs relative to each other. As usedherein, the term “CDR set” refers to the three hypervariable regions ofa heavy or light chain V region. Proceeding from the N-terminus of aheavy or light chain, these regions are denoted as “CDR1,” “CDR2,” and“CDR3” respectively. An antigen-binding site, therefore, includes sixCDRs, comprising the CDR set from each of a heavy and a light chain Vregion. A polypeptide comprising a single CDR, (e.g., a CDR1, CDR2 orCDR3) is referred to herein as a “molecular recognition unit.”Crystallographic analysis of a number of antigen-antibody complexes hasdemonstrated that the amino acid residues of CDRs form extensive contactwith bound antigen, wherein the most extensive antigen contact is withthe heavy chain CDR3. Thus, the molecular recognition units areprimarily responsible for the specificity of an antigen-binding site.

As used herein, the term “FR set” refers to the four flanking amino acidsequences which frame the CDRs of a CDR set of a heavy or light chain Vregion. Some FR residues may contact bound antigen; however, FRs areprimarily responsible for folding the V region into the antigen-bindingsite, particularly the FR residues directly adjacent to the CDRs. WithinFRs, certain amino residues and certain structural features are veryhighly conserved. In this regard, all V region sequences contain aninternal disulfide loop of around 90 amino acid residues. When the Vregions fold into a binding-site, the CDRs are displayed as projectingloop motifs which form an antigen-binding surface. It is generallyrecognized that there are conserved structural regions of FRs whichinfluence the folded shape of the CDR loops into certain “canonical”structures—regardless of the precise CDR amino acid sequence. Further,certain FR residues are known to participate in non-covalent interdomaincontacts which stabilize the interaction of the antibody heavy and lightchains.

The structures and locations of immunoglobulin CDRs and variable domainsmay be determined by reference to Kabat, E. A. et al., Sequences ofProteins of Immunological Interest. 4th Edition. US Department of Healthand Human Services. 1987, and updates thereof, now available on theInternet (immuno.bme.nwu.edu). Alternatively, CDRs may be determined byusing “IMGT®, the international ImMunoGeneTics information System®available at http://www.imgt.org (see, e.g., Lefranc, M.-P. et al.(1999) Nucleic Acids Res., 27:209-212; Ruiz, M. et al. (2000) NucleicAcids Res., 28:219-221; Lefranc, M.-P. (2001) Nucleic Acids Res.,29:207-209; Lefranc, M.-P. (2003) Nucleic Acids Res., 31:307-310;Lefranc, M.-P. et al. (2004) In Silico Biol., 5, 0006 [Epub], 5:45-60(2005)]; Lefranc, M.-P. et al. (2005) Nucleic Acids Res., 33:D593-597;Lefranc, M.-P. et al. (2009) Nucleic Acids Res., 37:D1006-1012; Lefranc,M.-P. et al. (2015) Nucleic Acids Res., 43: D413-422).

A “monoclonal antibody” refers to a homogeneous antibody populationwherein the monoclonal antibody is comprised of amino acids (naturallyoccurring and non-naturally occurring) that are involved in theselective binding of an epitope. Monoclonal antibodies are highlyspecific, being directed against a single epitope. The term “monoclonalantibody” encompasses not only intact monoclonal antibodies andfull-length monoclonal antibodies, but also fragments thereof (such asFab, Fab′, F(ab′)2, Fv), single chain (scFv), VHH or sdAb, variantsthereof, fusion proteins comprising an antigen-binding fragment of amonoclonal antibody, humanized monoclonal antibodies, chimericmonoclonal antibodies, and any other modified configuration of theimmunoglobulin molecule that comprises an antigen-binding fragment(epitope recognition site) of the required specificity and the abilityto bind to an epitope, including Wnt surrogate molecules disclosedherein. It is not intended to be limited as regards the source of theantibody or the manner in which it is made (e.g., by hybridoma, phageselection, recombinant expression, transgenic animals, etc.). The termincludes whole immunoglobulins as well as the fragments etc. describedabove under the definition of “antibody”.

The proteolytic enzyme papain preferentially cleaves IgG molecules toyield several fragments, two of which (the F(ab) fragments) eachcomprise a covalent heterodimer that includes an intact antigen-bindingsite. The enzyme pepsin is able to cleave IgG molecules to provideseveral fragments, including the F(ab′)2 fragment which comprises bothantigen-binding sites. An Fv fragment for use according to certainembodiments of the present invention can be produced by preferentialproteolytic cleavage of an IgM, and on rare occasions of an IgG or IgAimmunoglobulin molecule. Fv fragments are, however, more commonlyderived using recombinant techniques known in the art. The Fv fragmentincludes a non-covalent V_(H)::V_(L) heterodimer including anantigen-binding site which retains much of the antigen recognition andbinding capabilities of the native antibody molecule. Inbar et al.(1972) Proc. Nat. Acad. Sci. USA 69:2659-2662; Hochman et al. (1976)Biochem 15:2706-2710; and Ehrlich et al. (1980) Biochem 19:4091-4096.

In certain embodiments, single chain Fv or scFV antibodies arecontemplated. For example, Kappa bodies (III et al., Prot. Eng. 10:949-57 (1997); minibodies (Martin et al., EMBO J 13: 5305-9 (1994);diabodies (Holliger et al., PNAS 90: 6444-8 (1993); or Janusins(Traunecker et al., EMBO J 10: 3655-59 (1991) and Traunecker et al.,Int. J. Cancer Suppl. 7: 51-52 (1992), may be prepared using standardmolecular biology techniques following the teachings of the presentapplication with regard to selecting antibodies having the desiredspecificity. In still other embodiments, bispecific or chimericantibodies may be made that encompass the ligands of the presentdisclosure. For example, a chimeric antibody may comprise CDRs andframework regions from different antibodies, while bispecific antibodiesmay be generated that bind specifically to one or more Fzd receptorthrough one binding domain and to a second molecule through a secondbinding domain. These antibodies may be produced through recombinantmolecular biological techniques or may be physically conjugatedtogether.

A single chain Fv (scFv) polypeptide is a covalently linked V_(H)::V_(L)heterodimer which is expressed from a gene fusion including V_(H)- andV_(L)-encoding genes linked by a peptide-encoding linker. Huston et al.(1988) Proc. Nat. Acad. Sci. USA 85(16):5879-5883. A number of methodshave been described to discern chemical structures for converting thenaturally aggregated—but chemically separated—light and heavypolypeptide chains from an antibody V region into an scFv molecule whichwill fold into a three dimensional structure substantially similar tothe structure of an antigen-binding site. See, e.g., U.S. Pat. Nos.5,091,513 and 5,132,405, to Huston et al.; and U.S. Pat. No. 4,946,778,to Ladner et al.

In certain embodiments, an antibody as described herein is in the formof a diabody. Diabodies are multimers of polypeptides, each polypeptidecomprising a first domain comprising a binding region of animmunoglobulin light chain and a second domain comprising a bindingregion of an immunoglobulin heavy chain, the two domains being linked(e.g. by a peptide linker) but unable to associate with each other toform an antigen binding site: antigen binding sites are formed by theassociation of the first domain of one polypeptide within the multimerwith the second domain of another polypeptide within the multimer(WO94/13804).

A dAb fragment of an antibody consists of a V_(H) domain (Ward, E. S. etal., Nature 341, 544-546 (1989)).

Where bispecific antibodies are to be used, these may be conventionalbispecific antibodies, which can be manufactured in a variety of ways(Holliger, P. and Winter G. Current Opinion Biotechnol. 4, 446-449(1993)), e.g. prepared chemically or from hybrid hybridomas, or may beany of the bispecific antibody fragments mentioned above. Diabodies andscFv can be constructed without an Fc region, using only variabledomains, potentially reducing the effects of anti-idiotypic reaction.

Bispecific diabodies, as opposed to bispecific whole antibodies, mayalso be particularly useful because they can be readily constructed andexpressed in E. coli. Diabodies (and many other polypeptides such asantibody fragments) of appropriate binding specificities can be readilyselected using phage display (WO94/13804) from libraries. If one arm ofthe diabody is to be kept constant, for instance, with a specificitydirected against antigen X, then a library can be made where the otherarm is varied and an antibody of appropriate specificity selected.Bispecific whole antibodies may be made by knobs-into-holes engineering(J. B. B. Ridgeway et al., Protein Eng., 9, 616-621, 1996).

In certain embodiments, the antibodies described herein may be providedin the form of a UniBody®. A UniBody® is an IgG4 antibody with the hingeregion removed (see GenMab Utrecht, The Netherlands; see also, e.g.,US20090226421). This proprietary antibody technology creates a stable,smaller antibody format with an anticipated longer therapeutic windowthan current small antibody formats. IgG4 antibodies are consideredinert and thus do not interact with the immune system. Fully human IgG4antibodies may be modified by eliminating the hinge region of theantibody to obtain half-molecule fragments having distinct stabilityproperties relative to the corresponding intact IgG4 (GenMab, Utrecht).Halving the IgG4 molecule leaves only one area on the UniBody® that canbind to cognate antigens (e.g., disease targets) and the UniBody®therefore binds univalently to only one site on target cells.

In certain embodiments, the antibodies of the present disclosure maytake the form of a VHH or sdAb. VHH or sdAb technology was originallydeveloped following the discovery and identification that camelidae(e.g., camels and llamas) possess fully functional antibodies thatconsist of heavy chains only and therefore lack light chains. Theseheavy-chain only antibodies contain a single variable domain (V_(HH))and two constant domains (C_(H)2, C_(H)3). The cloned and isolatedsingle variable domains have full antigen binding capacity and are verystable. These single variable domains, with their unique structural andfunctional properties, form the basis of “VHH or sdAb”. VHH or sdAb areencoded by single genes and are efficiently produced in almost allprokaryotic and eukaryotic hosts e.g. E. coli (see e.g. U.S. Pat. No.6,765,087), molds (for example Aspergillus or Trichoderma) and yeast(for example Saccharomyces, Kluyvermyces, Hansenula or Pichia (see e.g.U.S. Pat. No. 6,838,254). The production process is scalable andmulti-kilogram quantities of VHH or sdAb have been produced. VHH or sdAbmay be formulated as a ready-to-use solution having a long shelf life.The VHH or sdAb method (see, e.g., WO 06/079372) is a proprietary methodfor generating VHH or sdAb against a desired target, based on automatedhigh-throughput selection of B-cells. VHH or sdAb are single-domainantigen-binding fragments of camelid-specific heavy-chain onlyantibodies. VHH antibodies or sdAb, typically have a small size ofaround 15 kDa.

In certain embodiments, the antibodies or antigen-binding fragmentsthereof as disclosed herein are humanized. This refers to a chimericmolecule, generally prepared using recombinant techniques, having anantigen-binding site derived from an immunoglobulin from a non-humanspecies and the remaining immunoglobulin structure of the molecule basedupon the structure and/or sequence of a human immunoglobulin. Theantigen-binding site may comprise either complete variable domains fusedonto constant domains or only the CDRs grafted onto appropriateframework regions in the variable domains. Epitope binding sites may bewild type or modified by one or more amino acid substitutions. Thiseliminates the constant region as an immunogen in human individuals, butthe possibility of an immune response to the foreign variable regionremains (LoBuglio, A. F. et al., (1989) Proc Natl Acad Sci USA86:4220-4224; Queen et al., PNAS (1988) 86:10029-10033; Riechmann etal., Nature (1988) 332:323-327). Illustrative methods for humanizationof the anti-Fzd antibodies disclosed herein include the methodsdescribed in U.S. Pat. No. 7,462,697.

Another approach focuses not only on providing human-derived constantregions, but modifying the variable regions as well so as to reshapethem as closely as possible to human form. It is known that the variableregions of both heavy and light chains contain threecomplementarity-determining regions (CDRs) which vary in response to theepitopes in question and determine binding capability, flanked by fourframework regions (FRs) which are relatively conserved in a givenspecies and which putatively provide a scaffolding for the CDRs. Whennonhuman antibodies are prepared with respect to a particular epitope,the variable regions can be “reshaped” or “humanized” by grafting CDRsderived from nonhuman antibody on the FRs present in the human antibodyto be modified. Application of this approach to various antibodies hasbeen reported by Sato, K., et al., (1993) Cancer Res 53:851-856.Riechmann, L., et al., (1988) Nature 332:323-327; Verhoeyen, M., et al.,(1988) Science 239:1534-1536; Kettleborough, C. A., et al., (1991)Protein Engineering 4:773-3783; Maeda, H., et al., (1991) HumanAntibodies Hybridoma 2:124-134; Gorman, S. D., et al., (1991) Proc NatlAcad Sci USA 88:4181-4185; Tempest, P. R., et al., (1991) Bio/Technology9:266-271; Co, M. S., et al., (1991) Proc Natl Acad Sci USA88:2869-2873; Carter, P., et al., (1992) Proc Natl Acad Sci USA89:4285-4289; and Co, M. S. et al., (1992) J Immunol 148:1149-1154. Insome embodiments, humanized antibodies preserve all CDR sequences (forexample, a humanized mouse antibody which contains all six CDRs from themouse antibodies). In other embodiments, humanized antibodies have oneor more CDRs (one, two, three, four, five, six) which are altered withrespect to the original antibody, which are also termed one or more CDRs“derived from” one or more CDRs from the original antibody.

In certain embodiments, the antibodies of the present disclosure may bechimeric antibodies. In this regard, a chimeric antibody is comprised ofan antigen-binding fragment of an antibody operably linked or otherwisefused to a heterologous Fc portion of a different antibody. In certainembodiments, the heterologous Fc domain is of human origin. In otherembodiments, the heterologous Fc domain may be from a different Ig classfrom the parent antibody, including IgA (including subclasses IgA1 andIgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4),and IgM. In further embodiments, the heterologous Fc domain may becomprised of CH2 and CH3 domains from one or more of the different Igclasses. As noted above with regard to humanized antibodies, theantigen-binding fragment of a chimeric antibody may comprise only one ormore of the CDRs of the antibodies described herein (e.g., 1, 2, 3, 4,5, or 6 CDRs of the antibodies described herein), or may comprise anentire variable domain (VL, VH or both).

Wnt Surrogates

The disclosure provides, in certain aspects, Wnt surrogate moleculesthat bind both one or more Fzd receptors and one or both of LRP5 and/orLRP6. Wnt surrogate molecules may also be referred to as “Wntsurrogates” or “Wnt mimetics.” In particular embodiments, the Wntsurrogate molecules bind one or more human Fzd receptors and one or bothof a human LRP5 and/or a human LRP6.

In certain embodiments, a Wnt surrogate molecule is capable ofmodulating or modulates Wnt signaling events in a cell contacted withthe Wnt surrogate molecule. In certain embodiments, the Wnt surrogatemolecule increases Wnt signaling, e.g., via the canonical Wnt/β-cateninpathway. In certain embodiments, the Wnt surrogate molecule specificallymodulates the biological activity of a human Wnt signaling pathway.

Wnt surrogate molecules of the present invention are biologically activein binding to one or more Fzd receptor and to one or more of LRP5 andLRP6, and in activation of Wnt signaling, i.e., the Wnt surrogatemolecule is a Wnt agonist. The term “Wnt agonist activity” refers to theability of an agonist to mimic the effect or activity of a Wnt proteinbinding to a frizzled protein and/or LRP5 or LRP6. The ability of theWnt surrogate molecules and other Wnt agonists disclosed herein to mimicthe activity of Wnt can be confirmed by a number of assays. Wnt agoniststypically initiate a reaction or activity that is similar to or the sameas that initiated by the receptor's natural ligand. In particular, theWnt agonists disclosed herein activate, enhance or increase thecanonical Wnt/β-catenin signaling pathway. As used herein, the term“enhances” refers to a measurable increase in the level of Wnt/β-cateninsignaling compared with the level in the absence of a Wnt agonist, e.g.,a Wnt surrogate molecule disclosed herein. In particular embodiments,the increase in the level of Wnt/β-catenin signaling is at least 10%, atleast 20%, at least 50%, at least two-fold, at least five-fold, at least10-fold, at least 20-fold, at least 50-fold, or at least 100-fold ascompared to the level of Wnt/β-catenin signaling in the absence of theWnt agonist, e.g., in the same cell type. Methods of measuringWnt/β-catenin signaling are known in the art and include those describedherein.

In particular embodiments, Wnt surrogate molecules disclosed herein arebispecific, i.e., they specifically bind to two or more differentepitopes, e.g., one or more Fzd receptor, and LRP5 and/or LRP6.

In particular embodiments, Wnt surrogate molecules disclosed herein aremultivalent, e.g., they comprise two or more regions that eachspecifically bind to the same epitope, e.g., two or more regions thatbind to an epitope within one or more Fzd receptor and/or two or moreregions that bind to an epitope within LRP5 and/or LRP6. In particularembodiments, they comprise two or more regions that bind to an epitopewithin one or more Fzd receptor and two or more regions that bind to anepitope within LRP5 and/or LRP6. In certain embodiments, Wnt surrogatemolecules comprise a ratio of the number of regions that bind one ormore Fzd receptor to the number of regions that bind LRP5 and/or LRP6 ofor about: 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 2:3, 2:5, 2:7, 7:2, 5:2, 3:2,3:4, 3:5, 3:7, 3:8, 8:3, 7:3, 5:3, 4:3, 4:5, 4:7, 4:9, 9:4, 7:4, 5:4,6:7, 7:6, 1:2, 1:3, 1:4, 1:5, or 1:6. In certain embodiments, Wntsurrogate molecules are bispecific and multivalent.

Wnt surrogate molecules disclosed herein may have any of a variety ofdifferent structural formats or configurations. Wnt surrogate moleculesmay comprise polypeptides and/or non-polypeptide binding moieties, e.g.,small molecules. In particular embodiments, Wnt surrogate moleculescomprise both a polypeptide region and a non-polypeptide binding moiety.In certain embodiments, Wnt surrogate molecules may comprise a singlepolypeptide, or they may comprise two or more, three or more, or four ormore polypeptides. In certain embodiments, one or more polypeptides of aWnt surrogate molecule are antibodies or antigen-binding fragmentsthereof. In certain embodiments, Wnt surrogates comprise two antibodiesor antigen binding fragments thereof, one that binds one or more Fzd andone that binds LRP5 and/or LRP6. In certain embodiments, the Wntsurrogates comprises one, two, three, or four polypeptides, e.g., linkedor bound to each other or fused to each other.

When the Wnt surrogate molecules comprise a single polypeptide, they maybe a fusion protein comprising one or more Fzd binding domain and one ormore LRP5/6 binding domain. The binding domains may be directly fused orthey may be connected via a linker, e.g., a polypeptide linker,including but not limited to any of those disclosed herein.

When the Wnt surrogate molecules comprise two or more polypeptides, thepolypeptides may be linked via covalent bonds, such as, e.g., disulfidebonds, and/or noncovalent interactions. For example, heavy chains ofhuman immunoglobulin IgG interact at the level of their CH3 domainsdirectly, whereas, at the level of their CH2 domains, they interact viathe carbohydrate attached to the asparagine (Asn) N84.4 in the DE turn.In particular embodiments, the Wnt surrogate molecules comprise one ormore regions derived from an antibody or antigen-binding fragmentthereof, e.g., antibody heavy chains or antibody light chains orfragments thereof. In certain embodiments, a Wnt surrogate polypeptidecomprises two antibody heavy chain regions (e.g., hinge regions) boundtogether via one or more disulfide bond. In certain embodiments, a Wntsurrogate polypeptide comprises an antibody light chain region (e.g., aCL region) and an antibody heavy chain region (e.g., a C_(H)1 region)bound together via one or more disulfide bond.

Wnt surrogate polypeptides may be engineered to facilitate bindingbetween two polypeptides. For example, Knob-into-holes amino acidmodifications may be introduced into two different polypeptides tofacilitate their binding. Knobs-into-holes amino acid (AA) changes is arational design strategy developed in antibody engineering, used forheterodimerization of the heavy chains, in the production of bispecificIgG antibodies. AA changes are engineered in order to create a knob onthe C_(H)3 of the heavy chains from a first antibody and a hole on theC_(H)3 of the heavy chains of a second antibody. The knob may berepresented by a tyrosine (Y) that belongs to the ‘very large’ IMGTvolume class of AA, whereas the hole may be represented by a threonine(T) that belongs to the ‘small’ IMGT volume class. Other means ofintroducing modifications into polypeptides to facilitate their bindingare known and available in the art. For example, specific amino acidsmay be introduced and used for cross-linking, such as Cysteine to forman intermolecular disulfide bond.

Wnt surrogate molecules may have a variety of different structuralformats, including but not limited to those shown in FIG. 1.

In one embodiment, a Wnt surrogate molecule comprises an scFv orantigen-binding fragment thereof fused to a VHH or sdAb orantigen-binding fragment thereof. In certain embodiments, the scFvspecifically binds one or more Fzd receptor, and the VHH or sdAbspecifically binds LRP5 and/or LRP6. In certain embodiments, the scFvspecifically binds LRP5 and/or LRP6, and the VHH or sdAb specificallybinds one or more Fzd receptor. In particular embodiments, the scFv orantigen-binding fragment thereof is fused directly to the VHH or sdAb orantigen-binding fragment thereof, whereas in other embodiments, the twobinding regions are fused via a linker moiety. In particularembodiments, the VHH or sdAb is fused to the N-terminus of the scFV,while in other embodiments, the VHH or sdAb is fused to the C-terminusof the scFv. In particular embodiments, the scFv is described herein orcomprises any of the CDR sets described herein. In particularembodiments, the VHH or sdAb is described herein or comprises any of theCDR sets disclosed herein.

In various embodiments, including but not limited to those depicted inFIG. 1A, a Wnt surrogate molecule comprises one or more Fab orantigen-binding fragment thereof and one or more VHH or sdAb orantigen-binding fragment thereof (or alternatively, one or more scFv orantigen-binding fragment thereof). In certain embodiments, the Fabspecifically binds one or more Fzd receptor, and the VHH or sdAb (orscFv) specifically binds LRP5 and/or LRP6. In certain embodiments, theFab specifically binds LRP5 and/or LRP6, and the VHH or sdAb (or scFv)specifically binds one or more Fzd receptor. In certain embodiments, theVHH or sdAb (or scFv) is fused to the N-terminus of the Fab, while insome embodiments, the VHH or sdAb (or scFv) is fused to the C-terminusof the Fab. In particular embodiments, the Fab is present in a full IgGformat, and the VHH or sdAb (or scFv) is fused to the N-terminus and/orC-terminus of the IgG light chain. In particular embodiments, the Fab ispresent in a full IgG format, and the VHH or sdAb (or scFv) is fused tothe N-terminus and/or C-terminus of the IgG heavy chain. In particularembodiments, two or more VHH or sdAb (or scFvs) are fused to the IgG atany combination of these locations.

Fabs may be converted into a full IgG format that includes both the Faband Fc fragments, for example, using genetic engineering to generate afusion polypeptide comprising the Fab fused to an Fc region, i.e., theFab is present in a full IgG format. The Fc region for the full IgGformat may be derived from any of a variety of different Fcs, includingbut not limited to, a wild-type or modified IgG1, IgG2, IgG3, IgG4 orother isotype, e.g., wild-type or modified human IgG1, human IgG2, humanIgG3, human IgG4, human IgG4Pro (comprising a mutation in core hingeregion that prevents the formation of IgG4 half molecules), human IgA,human IgE, human IgM, or the modified IgG1 referred to as IgG1 LALAPG.The L235A, P329G (LALA-PG) variant has been shown to eliminatecomplement binding and fixation as well as Fc-γ dependentantibody-dependent cell-mediated cytotoxity (ADCC) in both murine IgG2aand human IgG1. These LALA-PG substitutions allow a more accuratetranslation of results generated with an “effectorless” antibodyframework scaffold between mice and primates. In particular embodimentsof any of the IgG disclosed herein, the IgG comprises one or more of thefollowing amino acid substitutions: N297G, N297A, N297E, L234A, L235A,or P236G.

Non-limiting examples of bivalent and bispecific Wnt surrogate moleculesthat are bivalent towards both the one or more Fzd receptor and the LRP5and/or LRP6 are provided as the top four structures depicted in FIG. 1A,where the VHH or sdAb or scFv is depicted in white, and the Fab or IgGis depicted in black. As shown, the VHH or sdAb (or scFvs) may be fusedto the N-termini of both light chains, to the N-termini of both heavychains, to the C-termini of both light chains, or to the C-termini ofboth heavy chains. It is further contemplated, e.g., that VHH or sdAb(or scFvs) could be fused to both the N-termini and C-termini of theheavy and/or light chains, to the N-termini of the light chains and theheavy chains, to the C-termini of the heavy and light chains, to theN-termini of the heavy chains and C-termini of the light chains, or tothe C-termini of the heavy chains and the N-termini of the light chains.In other related embodiments, two or more VHH or sdAb (or scFvs) may befused together, optionally via a linker moiety, and fused to the Fab orIgG at one or more of these locations. In a related embodiment, the Wntsurrogate molecule has a Hetero-IgG format, whereas the Fab is presentas a half antibody, and one or more VHH or sdAb (or scFv) is fused toone or more of the N-terminus of the Fc, the N-terminus of the Fab, theC-terminus of the Fc, or the C-terminus of the Fab. A bispecific butmonovalent to each receptor version of this format is depicted at thebottom of FIG. 1A. In certain embodiments, the Fab or antigen-bindingfragment (or IgG) thereof is fused directly to the VHH or sdAb (or scFv)or antigen-binding fragment thereof, whereas in other embodiments, thebinding regions are fused via a linker moiety. In particularembodiments, the Fab is described herein or comprises any of the CDRsets described herein. In particular embodiments, the VHH or sdAb orscFv is described herein or comprises any of the CDR sets disclosedherein.

In various embodiments, including but not limited to those depicted inFIG. 1B, a Wnt surrogate molecule comprises one or more Fab orantigen-binding fragment thereof that binds one or more Fzd receptor andone or more Fab or antigen-binding fragment thereof that binds LRP5and/or LRP6. In certain embodiments, it comprises two Fab orantigen-binding fragments thereof that bind one or more Fzd receptorand/or two Fab or antigen-binding fragments thereof that bind LRP5and/or LRP6. In particular embodiments, one or more of the Fab ispresent in a full IgG format, and in certain embodiments, both Fab arepresent in a full IgG format. In certain embodiments, the Fab in fullIgG format specifically binds one or more Fzd receptor, and the otherFab specifically binds LRP5 and/or LRP6. In certain embodiments, the Fabspecifically binds one or more Fzd receptor, and the Fab in full IgGformat specifically binds LRP5 and/or LRP6. In certain embodiments, theFab specifically binds LRP5 and/or LRP6, and the Fab in full IgG formatspecifically binds one or more Fzd receptor. In certain embodiments, theFab is fused to the N-terminus of the IgG, e.g., to the heavy chain orlight chain N-terminus, optionally via a linker. In certain embodiments,the Fab is fused to the N-terminus of the heavy chain of the IgG and notfused to the light chain. In particular embodiments, the two heavychains can be fused together directly or via a linker. An example ofsuch a bispecific and bivalent with respect to both receptors is shownat the top of FIG. 1B. In other related embodiments, two or more VHH orsdAb may be fused together, optionally via a linker moiety, and fused tothe Fab or IgG at one or more of these locations. In a relatedembodiment, the Wnt surrogate molecule has a Hetero-IgG format, whereasone of the Fab is present as a half antibody, and the other Fab is fusedto one or more of the N-terminus of the Fc, the N-terminus of the Fab,or the C-terminus of the Fc. A bispecific but monovalent to eachreceptor version of this format is depicted at the bottom of FIG. 1B. Incertain embodiments, the Fab or antigen-binding fragment thereof isfused directly to the other Fab or IgG or antigen-binding fragmentthereof, whereas in other embodiments, the binding regions are fused viaa linker moiety. In particular embodiments, the one or both of the twoFabs are described herein or comprise any of the CDR sets describedherein.

In certain embodiments, Wnt surrogate molecules have a format asdescribed in PCT Application Publication No. WO2017/136820, e.g., aFabs-in-tandem IgG (FIT-IG) format. Shiyong Gong, Fang Ren, Danqing Wu,Xuan Wu & Chengbin Wu (2017). FIT-IG also include the formats disclosedin “Fabs-in-tandem immunoglobulin is a novel and versatile bispecificdesign for engaging multiple therapeutic targets” mAbs, 9:7, 1118-1128,DOI: 10.1080/19420862.2017.1345401. In certain embodiments, FIT-IGscombine the functions of two antibodies into one molecule byre-arranging the DNA sequences of two parental monoclonal antibodiesinto two or three constructs and co-expressing them in mammalian cells.Examples of FIT-IG formats and constructs are provided in FIGS. 1A and1B and FIGS. 2A and 2B of PCT Application Publication No. WO2017/136820.In certain embodiments, FIT-IGs require no Fc mutation; no scFvelements; and no linker or peptide connector. The Fab-domains in eacharm work “in tandem” forming a tetravalent bi-specific antibody withfour active and independent antigen binding sites that retain thebiological function of their parental antibodies In particularembodiments, Wnt surrogates comprises a Fab and an IgG. In certainembodiments, the Fab binder LC is fused to the HC of the IgG, e.g., by alinker of various length in between. In various embodiment, the Fabbinder HC can be fused or unfused to the LC of the IgG. A variation ofthis format has been called Fabs-in-tandem IgG (or FIT-Ig).

In particular embodiments, Wnt surrogate molecules comprise two or moreVHH or sdAb (or scFvs), including at least one that binds one or moreFzd receptor and at least one that binds LRP5 and/or LRP6. In certainembodiments, one of the binding regions is a VHH or sdAb and the otheris an scFv. Wnt memetic molecules comprising two or more VHH or sdAb (orscFvs) may be formatted in a variety of configurations, including butnot limited to those depicted in FIG. 1C. In certain bispecific,bivalent formats, two or more VHH or sdAb (or scFvs) are fused in tandemor fused to two different ends of an Fc, optionally via one or morelinkers. Where linkers are present, the linker and its length may be thesame or different between the VHH or sdAb (or scFv) and the other VHH orsdAb (or scFv), or between the VHH or sdAb and Fc. For example, incertain embodiments, the VHH or sdAb is fused to the N-terminus and/orC-terminus of the IgG heavy chain. In particular embodiments, two ormore VHH or sdAb are fused to the IgG at any combination of theselocations. Non-limiting examples of bivalent and bispecific Wntsurrogate molecules of this format are depicted as the top sevenstructures depicted in FIG. 1C, where the first VHH or sdAb is depictedin white, the Fc or IgG is depicted in black, and the second VHH or sdAbis depicted as light gray. In various embodiments, both VHH or sdAb maybe fused to the N-termini of the Fc, to the C-termini of the Fc, or oneor more VHH or sdAb may be fused to either or both of an N-terminus orC-terminus of the Fc. In a related embodiment, the Wnt surrogatemolecule has a Hetero-IgG format, whereas one VHH or sdAb is present asa half antibody, and the other is fused to the N-terminus of the Fc orthe C-terminus of the Fc. A bispecific but monovalent to each receptorversion of this format is depicted at the bottom of FIG. 1C. In certainembodiments, the VHH or sdAb is fused directly to the other VHH or sdAbwhereas in other embodiments, the binding regions are fused via a linkermoiety. In particular embodiments, the VHH or sdAb are described hereinor comprises any of the CDR sets described herein. In variousembodiments, any of these formats may comprise one or more scFvs inplace of one or more VHH or sdAb.

In certain embodiments, a Wnt surrogate molecule is formatted as adiabody. As shown in FIG. 1D, the binders against Fzd and LRP can alsobe linked together in a diabody (or DART) configuration. The diabody canalso be in a single chain configuration. If the diabody is fused to anFc, this will create a bivalent bispecific format. Without fusion to Fc,this would be a monovalent bispecific format. In certain embodiments; adiabody is a noncovalent dimer scFv fragment that consists of theheavy-chain variable (VH) and light-chain variable (VL) regionsconnected by a small peptide linker. Another form of diabody is asingle-chain (Fv)2 in which two scFv fragments are covalently linked toeach other.

As discussed, Wnt surrogate molecules, in various embodiments, compriseone or more antibodies or antigen-binding fragments thereof disclosedherein. Thus, in particular embodiments, a Wnt surrogate comprises twopolypeptides, wherein each polypeptide comprises an Nab or scFv thatbinds LRP5/6 and an Nab or scFv that binds one or more Wnts, optionallywherein one of the binding domains is an scFv and the other is an Nab.In certain embodiments, a Wnt surrogate comprises three polypeptides,wherein the first polypeptide comprises an antibody heavy chain and thesecond polypeptide comprises an antibody light chain, wherein theantibody heavy chain and light chain bind LRP5/6 or one or more Fzds,and wherein the third polypeptide comprises a VHH or sdAb fused to aheavy chain Fc region, wherein the VHH or sdAb binds to either LRP5/6 orone or more Fzds. In other embodiments, Wnt polypeptides comprise fourpolypeptides, including two heavy chain polypeptides and two light chainpolypeptides, wherein the two heavy chains and two light chains bindLRP5/6 or one or more Fzds, and further comprise one or more Nab or scFvfused to one or more of the heavy chains and/or light chains, whereinthe Nab or scFv binds to LRP5/6 or one or more Fzds. In anotherillustrative embodiment, a Wnt surrogate comprises at least fourpolypeptides, including two heavy chain polypeptides and two light chainpolypeptides that bind either LRP5/6 or one or more Fzds, wherein theWnt surrogate further comprises a Fab that binds either LRP5/6 or one ormore Fzds. For example, the Fab may comprise two polypeptides, eachfused to one of the two heavy chain polypeptides, and two polypeptides,each fused to one of the two light chain polypeptides, or it maycomprise two polypeptides each fused to one of the two heavy chainpolypeptides and two additional polypeptides, each bound to one of thetwo polypeptides fused to the heavy chain polypeptides, thus making asecond Fab. Other configurations may be used to produce the Wntsurrogates disclosed herein.

In particular embodiments, a Wnt surrogate molecule comprises a Fzdbinding region, e.g., an anti-Fzd antibody, or antigen-binding fragmentthereof, fused or bound to a polypeptide that specifically binds to oneor more Fzd receptor. In particular embodiments, the polypeptide thatspecifically binds to one or more Fzd receptor is an antibody orantigen-binding fragment thereof. If certain embodiments, it is anantibody or antigen-binding fragment thereof disclosed herein or in theU.S. provisional patent application Ser. No. 62/607,877, titled,“Anti-Frizzled antibodies and Methods of Use,” Attorney docket numberSRZN-004/000S, filed on Dec. 19, 2017, which is incorporated herein byreference in its entirety. In particular embodiments, the Fzd bindingdomain comprises the three heavy chain CDRs and/or the three light chainCDRs disclosed for any of the illustrative antibodies or fragmentsthereof that bind to one or more Fzd receptor provided in Table 1A. Inparticular embodiments, the Fzd binding domain comprises the three heavychain CDRs and/or the three light chain CDRs disclosed for any of theillustrative antibodies or fragments thereof that bind to one or moreFzd receptor provided in Table 1A, wherein the CDRs collectivelycomprise one, two, three, four, five, six, seven, or eight amino acidmodifications, e.g., substitutions, deletions, or additions. In certainembodiments, the Fzd binding domain is a VHH or sdAb or was derived froma VHH or sdAb, so Table 1A only includes the three heavy chain CDRs. Inparticular embodiments, the Fzd binding domain comprises the three CDRHC sequences provided in Table 1A or variants wherein the CDRscollectively comprise one, two, three, four, five, six, seven or eightamino acid modifications. In particular embodiments, the Fzd bindingdomain comprises the heavy chain fragment and/or light chain fragment ofany of the illustrative antibodies or fragments thereof that bind to oneor more Fzd receptor provided in Table 1B or SEQ ID NOs:1-65 or 129-132(or an antigen-binding fragment or variant of either). In certainembodiments, the Fzd binding domain is an Fab or was derived from anFab, so the heavy chain of Table 1B includes VH and CH1 sequence, butnot CH2 or CH3 sequences. In certain embodiments, the Fzd binding domainis a VHH or sdAb or was derived from a VHH or sdAb, so Table 1B includesthe VHH domain. In certain embodiments, the Fzd binding region is apolypeptide, e.g., an antibody or antigen-binding fragment thereof, thatcompetes with any of these antibodies for binding to one or more Fzdreceptor.

TABLE 1A Anti-Fzd Antibody clone IDs and CDR sequences CDRH1 CDRH2 CDRH3CDRL1 CDRL2 CDRL3 Clone Initial SEQ SEQ SEQ SEQ SEQ SEQ ID Binding CDRH1ID CDRH2 ID CDRH3 ID CDRL1 ID CDRL2 ID CDRL3 ID 00 Fzd YTFTS 391 GWIS570 CARASAW 752 SGSSSNI 1156 SNY 1256 CAAW 1265 1S 1 YGIS AYNG TPYGAFDIGSHTVS QRP DGSLF - NTNY W S GHWV A0 A T 1 00 Fzd GSISS 283 GSIYH 547CARFYYDIL 818 RSSRSLL 1142 TLS 1259 CMQSI 1295 1S 1 GGYS SGSTY TGYSYFDYDTDDGN HRA QLPW - WS YN W TYLD S TF B0 1 00 Fzd GSISN 282 GEIDR 488CARVRARR 945 SGNTLGS 1155 QDS 1246 CQVW 1431 1S 1 YYWS SGDT FLVSDRSAFHYVS KRP DSSTV - NYN DIW S VF E0 1 00 Fzd GSISG 281 GSIYF 546 CARVMLIT942 RSSQSLL 1138 LGS 1237 CMQG 1289 1S 1 NNYY TGGT DAFDIW HSNGYN NRATHWP - ZG YYN YLD S YTF F0 1 00 Fzd GSISS 285 GYIYY 589 CARATYGG 760TRSSSNI 1161 GNS 1220 CGTW 1267 1S 1 SSYY SGSTY GAGYDV IRPS DSSLS - WGYN DAFDIW AWVF G H 01 00 Fzd GSISS 284 GYIYY 589 CARHAGFY 875 RSSQSLL1138 LGS 1236 CMQA 1280 1S 1 GGYY SGSTY GLADYFDY HSNGYN KRA LQIPP - WSYN W YLD S TF H0 1 00 Fzd GSISS 284 GYIYY 589 CARGKGYS 845 QASQDI 1041DAS 1185 CQQN 1332 1S 1 GGYY SGSTY YGYGKDW GKYLN NLE DYLPL - WS YN FDPWT TF A0 2 00 Fzd GSISG 280 GSIYF 546 CARVMLIT 942 RSSQSLL 1138 LGS 1237CMQG 1289 1S 1 NNYY TGGT DAFDIW HSNGYN NRA THWP - WG YYN YLD S YTF E0 200 Fzd GAISG 266 GSIYY 548 CARIGIAVA 882 RASQSV 1110 DAS 1188 CMQA 12841S 1 TSYF TGNT APVDHW GTYLT NRA TQFPL - WG YYN T TF G 02 00 Fzd GSISS285 GYIYY 589 CARATYGG 760 TRSSSNI 1161 GNS 1220 CGTW 1267 1S 1 SSYYSGSTY DAFDIW GAGYDV IRPS DSSLS - WG YN H AWVF H0 2 00 Fzd GSISS 284GYIYY 589 CARVRDYY 946 RASRSISS 1128 AAS 1175 CQQA 1314 1S 1 GGYY SGSTYDSSGYYYD YFN SLQ DTFPP - WS YN YFDYW S TF A0 3 00 Fzd ASFS 158 GEID 487CARGGQG 835 SGDKVG 1154 EDS 1199 CQAW 1301 1S 1 GHY HTGS GYDWGHY HKYASQRP DSSTD - WT TNYE HGLDVW S VVF B0 3 00 Fzd RAFT 329 ATISG 466CAAASSLTS 678 1S 5 DNV GGGS TPYDLW - MA TFDD H0 8 00 Fzd RSFRT 333 AAIS422 CNTVTYTG 1005 1S 5 NALG WTG GSYKNYW - GSTY A0 YA 9 00 Fzd SIDSI 356AALTS 428 CNVITIVRG 1006 1S 5 NAM GGITY MGPRAYW - A HA B0 9 00 FzdSIFSIN 357 ATIQS 465 CNVITIVRG 1006 1S 5 AMG GGRT MGPRAYW - NYA C0 9 00Fzd YTFTS 391 GWIS 570 CARDGTPF 772 QGDSLRT 1052 GKN 1219 CNSRD 1300 1S8 YGIS AYNG YSGSYYGS YYAS NRP NSGK - NTNY W S HKVF C0 A 7 00 Fzd GTFSS295 GRIIPI 529 CARVPTSP 944 RSSQSLL 1136 FGS 1206 CMQN 1291 1S 8 YAISLGIAN YDILTGPFD HSNGFN YRA LQTP - YA YW YVD S WTF D0 7 00 Fzd ASVSS 159YRSK 542 CARWKNY 953 RASQGIR 1070 AAS 1177 CLQDY 1273 1S 8 NSAA WYN FDPWSDLA TLES SYPRT - WN DYA F E0 7 00 Fzd FTFSS 228 STISG 646 CAKDLVP 704RASQSVS 1121 GAS 1213 CQQY 1410 1S 8 YAMS GGGS WGSSAFNI SYLA SRA GSSPP -TYYA W T TF H0 7 00 Fzd FTFST 243 SGVS 618 CARGQSEK 856 RASQGIS 1076 AAS1165 CQQT 1394 4S 5 YEMN WNG WWSGLYG SALA ALQ YSTPR - SRTH MDVW S TF E0YV 5 00 Fzd GTFST 298 GWIN 565 CWTGLLW 1031 RASQSIS 1098 AAS 1175 CQQS1372 4S 5 YAIS SGNG FGESTDAF SYLN SLQ YSTPL - NTKY DIW S TF E0 S 3 00Fzd GTFTY 307 GGIIPI 501 CASSMVR 964 RASQSIS 1098 AAS 1175 CQQS 1372 4S5 RYLH FGTG VPYYYGM SYLN SLQ YSTPL - NYA DVW S TF G 06 00 Fzd GPFN 272AGISR 445 CASKTTINS 958 1S 8 LFAM TGGN GWSREYH - G TGYA YW D0 9 00 FzdGPFN 272 AGISR 445 CASKTTINS 958 1S 8 LFAM TGGN GWSREYH - G TGYA YW E0 900 Fzd GFFSS 268 AAISR 409 CNALAPGV 987 1S 8 FTMG NGVY RGSW - TRFA F0 900 Fzd SLFRL 360 ATIST 467 CTDEESW 1011 1S 8 NGM RGTT - G HYA G 09 00Fzd GPFN 273 AGISR 445 CASKTTINS 958 1S 8 LLAM TGGN GWSREYH - G TGYA YWH0 9 00 Fzd SVVN 364 AAITS 425 CNRVGSRE 1001 1S 8 FVVM GGST YSYW - G NYAA1 0 00 Fzd AAIG 352 YKVK 404 CNAVTYN 994 1S 8 RTSDL WNG GYTIW - YTMGERTYY B1 L 0 00 Fzd SIFSS 359 ALITT 455 CNAGAPA 986 1S 1 NTIY SGNTWTYRMGT - NYA YYPQFGS G W 12 00 Fzd STFST 362 AAISG 408 CVKFGMN 1028 2S1 YAM SGEN LGYSGYDY - G TYYA W A0 1 00 Fzd STFSN 361 AAIS 411 CAAGPIAR681 2S 1 YAM WGG WYRGDM - G GSTFY DYW B0 S 1 00 Fzd RMFS 331 AAISS 410CAAGPIAR 681 2S 1 NYA GGSG WYRGDM - MG TYYS DYW C0 1 00 Fzd RTDG 337ATVT 469 CAAGPIAR 681 2S 1 GYVM WRTG WYRGDM - G TTYY DYW D0 A 1 00 FzdRTFSS 345 AAIS 421 CATLTPYG 974 2S 1 AAM WSGS TVASY - G TAYY E0 A 1 00Fzd RTFSS 347 AAVN 430 CAAVFLSR 689 2S 1 YAM WSG NYEIQEYY - G GSTY RYQF0 YA 1 00 Fzd RTFSS 347 AAIS 418 CAAGPIAR 681 2S 1 YAM WSG WYRGDM - GGSTY DYW G YA 01 00 Fzd RSFST 336 TVISG 676 CAAGPIAR 681 2S 1 YPMG SGGSWYRGDM - TYYS DYW H0 1 00 2S Fzd RRFTT 332 AAVT 436 CYLEGPLD 1032 - 1YGM WRSG VYW A0 G STYYA 2 00 Fzd RTFN 341 AAIS 415 CAKLGGSS 724 2S 1 RHVWSG WLREYDY - MG DSTY W B0 YA 2 00 Fzd RTFR 342 SAIS 603 CAAGPIAR 681 2S1 AYAM WSG WYRGDM - G GSTY DYW C0 YA 2 00 Fzd RTFSE 343 AAIS 417CNADSLRG 984 2S 1 YAM WSG IDYW - G GSTH D0 YA 2 00 Fzd FTFRE 199 SGISR613 CAPRVLVT 734 2S 1 YAMT DGGR APSGGMD - TSYS YW E0 2 00 Fzd GDFT 267AAVN 429 CAAVFLSR 689 2S 1 NYA WRG NYEIQEYY - MA DGTY RYQ F0 YS 2 00 FzdRTFG 340 AAISY 424 CAAGPIAR 681 2S 1 TWA NGFS WYRGDM - MG TYYS DYW G 0200 Fzd RTFSS 347 AAIS 418 CAAGPIAR 681 2S 1 YAM WSG WYRGDM - G GSTY DYWH0 YA 2 00 Fzd RTFGS 339 AAIS 418 CAAGPIAR 681 2S 1 YAM WSG WYRGDM - GGSTY DYW D0 YA 3 00 Fzd SIFSIY 358 AVVA 481 CNMRGN 1000 2S 1 AMG TGGAWYREGRP - TNYA AEFLSW E0 3 00 Fzd RTSSS 353 AAIS 418 CAAGPIAR 681 2S 1YAM WSG WYRGDM - G GSTY DYW F0 YA 3 00 Fzd RTFGS 339 AAIS 418 CAAGPIAR681 2S 1 YAM WSG WYRGDM - G GSTY DYW G YA 03 00 Fzd QTFT 327 AAIS 420CNAWVLV 996 2S 1 AYAM WSGS AGSRGTSA - G ATHY DYW H0 A 3 00 Fzd RTFSS 347AAIS 419 CAAGPIAR 681 2S 1 YAM WSGR WYRGDM - G STYYA DYW A0 4 00 FzdRTFSS 347 AAIS 418 CAAGPNYS 682 2S 1 YAM WSG WFMPSSS - G GSTY RLIW B0 YA4 00 Fzd RRFTT 332 AAVT 435 CSADKLDY 1010 2S 1 YGM WRA LDDQPFKT - G GSTYWDYW C0 YA 4 00 Fzd GTSST 309 AAIN 405 CAAVFLSR 689 2S 1 YAM RSGGNYEIQEYY - G STYYA RYQ D0 4 00 Fzd GTFST 300 AAIS 416 CAAGPIAR 681 2S 1YAM WSG WYRGDM - G DSTY DYW E0 YL 4 00 Fzd GTFSS 295 GWIS 577 CARGGAG826 RASQSIS 1098 AAS 1175 CQQS 1372 4S 5 YAIS TYNG RFGEGMD SYLN SLQYSTPL - ATNY VW S TF H0 A 4 00 Fzd YTFTS 391 GWIS 577 CASSKEKA 963GLSSGSV 1035 YTN 1263 CLLYL 1271 1S 5 YGIS AYNG TYYYGMD STNYYPS TRSGRGI - NTNY VW S WVF A0 A 4 00 Fzd GTFSS 295 GRIIPI 529 CARLDPGY 886TGTSSDV 1159 DVT 1196 CFSYA 1266 1S 5 YAIS LGIAN YYGMDVW GGYNSV KRPGSRF - YA S S D0 3 00 Fzd GTFSS 295 GGIIPI 499 CARVIFSTV 939 TRSSGSI1160 END 1202 CQSYD 1430 1S 5 YAIS FGTA TTTNDIW ASNYVQ KRP YDHR - NYA SWVF F0 3 00 Fzd YTFSG 374 GTVT 549 CARVDGSG 933 RASQSIS 1098 AAS 1175CQQS 1372 4S 5 YYLH PILGT YYGIDYW SYLN SLQ YSTPL - ANYA S TF E0 4 00 FzdGSFS 278 GRIIPI 530 CARTYLKA 930 RASQSIS 1098 AAS 1175 CQQS 1372 4S 5NYAIS LGSA FDIW SYLN SLQ YSTPL - NYA S TF A0 6 00 Fzd YTFTN 383 GRIN 537CARDRFDN 788 RASQGIS 1076 AAS 1179 CQQS 1351 4S 5 NFMH PNSG WFDPW SALATLQ YNTP - GTNY S WTF F0 A 4 00 Fzd GTFSS 295 GRIIPI 529 CAREGRSR 808RSSQSLL 1139 MG 1238 CMHG 1279 1S 5 YAIS LGIAN VYGGNSFD RRNGHN SNRLHPPF - YA YW YVD AP TF C0 3 00 Fzd YIFTD 368 GGIIPI 499 CARMSSDY 895RASQGIS 1072 GAS 1215 CQQA 1312 3S 1 YYMH FGTA YDSSGYYR NNLN TLQ DSFPP -NYA RGMDVW S TF A0 1 00 Fzd YIFTD 368 GGIIPI 499 CARAWKG 761 RASQSIS1098 AAS 1175 CQQS 1372 3S 1 YYMH FGTA LWFGEGTF SYLN SLQ YSTPL - NYA DYWS TF E0 1 00 Fzd GTFSS 295 GWIN 558 CARLAFDI 885 RASQSIS 1098 AAS 1175CQQS 1372 3S 1 YAIS AGNG W SYLN SLQ YSTPL - NTTY S TF F0 A 1 00 FzdYTFTG 379 GWIN 558 CAKDRGN 707 RASQGIS 1074 EVS 1204 CQQS 1370 3S 1 YYMHAGNG YGDYLDY NYLA SVQ YSTPL - NTTY W G AF A0 A 2 00 Fzd FTFSN 214 ALISY454 CTRGSRIG 1015 RASQSIG 1084 AAS 1171 CQQG 1325 3S 1 SDM DGSH WFDPWRWLA RLQ FNFPL - N TYYA S TF C0 2 00 Fzd GTFSS 296 GGIIPI 505 CARARGG749 RASQGIS 1072 TAS 1258 CLQDY 1274 3S 1 YTIS SGKT DSPLSL NNLN SLQSYPYT - DYA S F E0 2 00 Fzd GTFRS 292 GGIIPI 499 CARGGWR 840 RASQSVS1115 GAS 1217 CQQY 1405 3S 1 YAIN FGTA PDYYGSGS SDLA TRA ETWP - NYAYYSFDYW T VLTF F0 2 00 Fzd FTFGT 196 SGITG 616 CARMKDW 894 RASESVS 1056GAS 1217 CQQY 1420 3S 1 YWVT SGGR FGAFDIW SSSFA TRA NNWP - TFYA T PNYTFG 02 00 Fzd FTFSR 220 SYISG 658 CARGLVIA 849 QANQDI 1038 AAS 1175 CQQT1389 3S 1 YAMS DSGY TNWFDPW SNYLN SLQ YNPPR - TNYA S TF C0 3 00 FzdYTFTS 392 GWIN 567 CAESLTSTA 691 RASQGIS 1072 AAS 1174 CQQS 1368 3S 1YYMH TYNG DW NNLN SLQ YSTPF - NTNY R TF D0 P 3 00 Fzd YIFTD 368 NPTT 586CARNVEGA 898 RASQGIS 1072 SAS 1252 CQQS 1364 3S 1 YYMH GNTG TSFPEFDYNNLN NLQ YSPPP - YA W S YTF E0 3 00 Fzd GTFSS 295 GGIIPI 499 CAKDIGSS701 RASQSIS 1098 AAS 1175 CQQS 1372 3S 1 YAIS FGTA WYYYMDV SYLN SLQYSTPL - NYA W S TF H0 3 00 Fzd FTFGT 196 SGITG 616 CARMKDW 894 RTSERSSI1148 GAS 1217 CQQY 1420 3S 1 YWVT SGGR FGAFDIW SSFA TRA NNWP - TFYA TRNYTF A0 4 00 Fzd FAVSS 168 ASIW 463 CAPNESGN 733 RASQGIS 1072 KAS 1225CQQS 1339 3S 1 SYMS FDGS VDYW NNLN SLE YSTPH - NQDY N TF C0 A 4 00 FzdFTFSS 227 SAISG 600 CARDHGSS 774 QASQDI 1042 DVS 1195 CQHL 1304 3S 1 YAMSGGS WYQNTDA GNYLN NLE NSYPP - H TYYA FDIW R GDTF D0 4 00 Fzd FRFIS 177GRVIP 545 CASSSDYG 966 RASQSIS 1098 AAS 1175 CQQS 1372 3S 1 HPIH ILGVTDYLKEPNY SYLN SLQ YSTPL - NYA GMDVW S TF G 04 00 Fzd FTFSN 216 SAIGT 595CATAYRRP 969 RSSQSLL 1134 LGS 1237 CMQN 1293 3S 2 YAMT GGGT GGLDVWHSDGKTY NRA THWP - YYA LY S LTR D0 5 00 Fzd FTFSS 236 GRIKS 535 CARGSSSW863 KSSQSLL 1036 LGS 1237 CMQN 1293 3S 2 YTMS KANG YDW HSDGKTY NRATHWP - GTTD LY S LTR E0 YA 5 00 Fzd FTFA 188 SYISS 659 CARGTFD 865RASQGIS 1072 AAS 1170 CQQS 1372 3S 2 DYG GSYTI WLLSPSYD NNLN RLESYSTPL - MH YYS YW TF A0 6 00 Fzd FTFSN 217 SAISN 601 CTSSFLTGS 1018RASQDIS 1065 AAS 1175 CQQS 1353 3S 2 YGM SGGS QPSGYW SYLA SLQ YRTPL - HTYYA S TF C0 6 00 Fzd FTFSD 207 SSTSG 642 CARHNPGY 877 RASQSVS 1116 DAS1188 CQHR 1307 3S 2 YGM SGGN MGYYYGM SNLA NRA TSWPL - H SKYS DVW T TF G06 00 Fzd GTFSS 296 GLVD 520 CTILPAAAA 1012 RASQRV 1083 DAS 1184 CQQY1415 3S 2 YTIS PEDG GTYYYYG GNNLA IRAT KDWP - ETIYA MDVW TF H0 6 00 FzdFTFSD 205 SSITR 639 CARDGGY 768 RASQSV 1109 GSS 1221 CQQY 1414 3S 2 HYMSTPSG W GSYLA NRA GTSLL - GTTE A TF B0 YA 7 00 Fzd YTFTN 383 GIINP 513RRYCSSTSC 759 QASQGIS 1049 LGS 1233 CQQS 1368 3S 2 NFMH SGGS YPRDAFDINNLN DRA YSTPF - TSYA W S TF D0 7 00 Fzd YTFTN 383 GWIN 563 CARSVGEV 926RASQSIS 1098 AAS 1175 CQQS 1368 3S 2 NFMH PNSG GATMLGIG SYLN SLQ YSTPF -GTKY VWYWFDP S TF E0 A W 7 00 Fzd FTFSN 216 SAIGT 595 CATAYRRP 969RSSQSLL 1134 LGF 1232 CMQN 1293 3S 2 YAMT GGGT GGLDVW HSDGKTY NRA THWP -YYA LY S LTR A0 8 00 Fzd LTVST 324 AGIG 440 CARDLVAA 782 RASQGIR 1069GAS 1214 CQQS 1374 3S 2 NFMS WDST RPSNWDY NDLG TLQ YSTPR - NIGY W R VTFC0 A 8 00 Fzd FTFR 201 STISG 647 CARGGGYS 829 RSSRSLL 1143 LGS 1237 CMQS1298 3S 2 NSAM SGGS SSW HSDGKTY NRA SHWP - H TYYS LY S KTF E0 8 00 FzdFTFD 194 SAIGA 593 CASPTVTR 960 RASQSIS 1098 AAS 1175 CQQS 1372 3S 4 HNPGGGT R SYLN SLQ YSTPL - MN YYA S TF G 09 00 Fzd GTFSS 295 GWIN 558CARHYYGS 880 RASQSIS 1098 AAS 1175 CQQS 1372 3S 4 YAIS AGNG GSYPDW SYLNSLQ YSTPL - NTTY S TF C1 A 0 00 Fzd FNFGI 172 SYISG 658 CARVGPG 936RASQGIS 1076 AAS 1167 CQQT 1396 3S 4 YSMT DSGY GWFDPW SYLA NLL YSTP -TNYA G WTF D1 0 00 Fzd FTFSS 227 AGISA 442 CARPSTTG 901 RASQSIG 1085 AAS1178 CQQS 1380 3S 4 YAM SGGS TKAFDIW SNLD TLET YSVPD - H TYYA TF E1 0 00Fzd GTFSS 295 GWIN 558 CARHYYGS 880 RASQSIS 1103 ZAS 1264 CQQS 1372 3S 4YAIS AGNG GSYPDW ZYZN SLQ YSTPL - NTTY S TF A1 A 1 00 Fzd GTFSS 295GRIIPI 528 CARGARLD 820 RASQSIS 1098 AAS 1175 CQQS 1372 3S 4 YAIS FGTVYW SYLN SLQ YSTPL - NYA S TF G 11 00 Fzd YTFTG 379 GGIIPI 502 CASTDPSS967 RASQSIG 1085 DAS 1189 CQQSF 1341 3S 4 YYMH FGTP GLDYW SNLD SLESIMPLT - HYA F H1 1 00 Fzd GTFSS 295 GWIN 564 CARGGSSD 838 RASQSIS 1098AAS 1175 CQQS 1372 3S 4 YAIS PNSG VR SYLN SLQ YSTPL - GTNY S TF C1 A 200 Fzd FTFSS 227 SVIST 652 CARGGSSD 838 RASQSIS 1098 AAS 1175 CQQS 13723S 4 YAM SGDT VR SYLN SLQ YSTPL - H VLYT S TF F1 2 00 Fzd GTFSS 295GIINP 513 CAKDGVV 698 RAIQSISS 1054 AAS 1175 CQQS 1372 4S 4 YAIS SGGS RYLN SLQ YSTPL - TSYA S TF B0 1 00 Fzd FTFSN 213 STISS 650 CARASRID 754RASQDIR 1062 AAS 1179 CQQA 1311 4S 4 HYTS SGGR GGWPIIDH DELA TLQ DSFPL -TFYA L S TF C0 1 00 Fzd FTFTN 248 SAISG 600 CARATGFG 757 RASQSIS 1098AAS 1175 CQQS 1372 4S 4 YAMS SGGS TVVFDYW SYLN SLQ YSTPL - TYYA S TF D01 00 Fzd GTFSS 295 GWIN 558 CARHYYGS 880 ZACLRIIS 1163 FAS 1205 CQQS1372 4S 4 YAIS AGNG GSYPDW YLN SLQ YSTPL - NTTY S TF E0 A 1 00 Fzd GTFSS295 GWIN 558 CARDGVE 773 RASQGIS 1073 DAS 1190 CQQS 1344 4F 4 YAIS AGNGNWLA SLQ HITPY - NTTY S TF F0 A 1 00 Fzd FTFSN 215 ALMS 456 CAKGIVGD 717RASQSIS 1098 AAS 1175 CQQS 1372 4S 4 YAM PDGTI YGAFDIW SYLN SLQ YSTPL -H IYYA S TF H0 1 00 Fzd FTFSS 230 SSINN 630 CAKDHLAV 700 RASQSIS 1098AAS 1175 CQQS 1372 4S 4 YGM SSRTV ADAHGR SYLN SLQ YSTPL - H FYA S TF B02 00 Fzd FTFSS 227 AVISY 474 CAGGEVYE 692 RASQSIS 1098 AAS 1175 CQQS1372 4S 4 YAM DGSN L SYLN SLQ YSTPL - H EYYA S TF E0 2 00 Fzd FTFST 242AVISS 473 CAAPDVVV 685 RASQGIS 1076 AAS 1179 CQQA 1322 4S 4 YAM DGNNTADGYYW SALA TLQ NTVPF - H KYYT S TF F0 2 00 Fzd FTFA 190 ALISY 453CAKTLVTS 728 RASQSIS 1098 AAS 1175 CQQS 1372 4S 4 NYA DGGT HALHIW SYLNSLQ YSTPL - MN KYYA S TF G 02 00 Fzd FTFA 189 ALISY 452 CAKTLVTS 728RASQSIS 1098 AAS 1175 CQQS 1372 4S 4 NYA DGGN HALHIW SYLN SLQ YSTPL - MHKYYA S TF H0 2 00 Fzd GSFS 276 GEIN 489 CARGRRLV 858 TGSSSNI 1158 SDR1255 CQSYD 1429 1S 5 GYYW HSGS RFTVTSAF GAGFGV NRP SSLRA - H TNYN DIW HS SVF E0 3 00 Fzd GTFSS 295 GGIIPI 504 CARIPKPR 883 RSSQSLL 1137 LGS1234 CMQS 1297 5 YAIS LGIAN GYSYGDN HSNGNT DRT LQTPY - YA GSW YLD S TFB0 5 00 Fzd GNFK 271 GRIIP 525 CARQYCSG 908 RASQDIR 1063 QAS 1245 CQQS1361 4S 6 NYGIT ALGT GSCYPDAF SALA SLIS YSMP - ANYA DIR QTF A0 7 00 FzdFTFSS 233 GVISK 553 CASSRDGY 965 QASQDIR 1043 AAS 1175 CQQSS 1347 4S 6YSMN DGDN NRLAFDIW NYLN SLQ RFWT - KYYA S F B0 7 00 Fzd GTFSS 295 GRIIPI529 CARDGGD 767 RASQSIS 1098 AAS 1175 CQQS 1372 4S 6 YAIS LGIAN YGMDVWSYLN SLQ YSTPL - YA S TF A0 8 00 Fzd YTFTN 383 PNSG 537 CASQNYYG 961RASQSIS 1098 AAS 1175 CQQS 1372 4S 6 NFMH GTNY SGSYPGFD SYLN SLQ YSTPL -A YW S TF B0 8 00 Fzd YTFTY 394 GGIIPI 499 CATHDSSG 973 RASQSIS 1098 AAS1175 CQQS 1372 4S 6 RYLH FGTA YYSFDYW SYLN SLQ YSTPL - NYA S TF D0 8 00Fzd FSVSS 187 SAIGT 595 CTTRTYDSS 1024 RSSRSLL 1144 LGS 1237 CVQTT 14344S 6 NYM GGGT GYYETQNY HSNGNT NRA QSPLT - N YYA YMDVW YLQ S F E0 8 00Fzd FTFSD 208 AAISY 423 CARSAVAG 916 RASQSIS 1098 AAS 1175 CQQS 1372 4S6 YYMS DESN AFDIW SYLN SLQ YSTPL - KFYA S TF G 08 00 Fzd FTFRD 198 SGIS615 CARRSGYS 913 RASQSIS 1098 AAS 1175 CQQS 1372 4S 6 YAM WNS GSVYYYYGSYLN SLQ YSTPL - N GSIGY MDVW S TF A0 A 9 00 Fzd FTFSS 221 AGIN 441CARGPSHQ 854 RASQGIS 1076 AAS 1175 CQQS 1357 4S 6 FGMH WNG HTFDIW SALASLQ YSHTA - GSVV S FTF B0 YA 9 00 Fzd YTFTN 383 GGFD 492 CARVGRGY 937RASQSIS 1098 AAS 1175 CQQS 1372 4S 6 NFMH PEDG SFDYW SYLN SLQ YSTPL -ETIYA S TF C0 9 00 Fzd DTFS 163 GRISA 538 CARSSGYV 924 RASQSIS 1098 AAS1175 CQQS 1372 4S 6 NYVIS YNGY GWFDPW SYLN SLQ YSTPL - KSYA S TF E0 9 00Fzd FTFSN 218 SYISG 657 CARLPRRS 888 RASQSIS 1098 AAS 1175 CQQS 1372 4S6 YYTS AGGS GKGSAFDI SYLN SLQ YSTPL - TEYA W S TF F0 9 00 Fzd GTFSS 296NPNS 583 CARVGATS 935 RASQSIS 1098 AAS 1175 CQQS 1372 4S 6 YTIS GNTGAGGMDV SYLN SLQ YSTPL - YA W S TF H0 9 00 Fzd YIFTD 368 GLVD 520CAHSDFFS 693 RASQSIS 1098 AAS 1175 CQQS 1372 4S 6 YYMH PEDG GLSFGDW SYLNSLQ YSTPL - ETIYA S TF C1 0 00 Fzd FTFSN 214 SSIST 637 CARGSYW 864RASQNIN 1081 RAS 1249 CQQY 1425 4S 6 SDM SGGS NYLA TLQ SSYPY - N TYYA STI D1 0 00 Fzd TTLNK 365 GRITP 539 CALSSSWY 731 RASQSIS 1098 AAS 1175CQQS 1372 4S 6 YAIS VVGV GGFDYW SYLN SLQ YSTPL - TNYA S TF E1 0 00 FzdGFTFS 269 ALVG 458 CNTGIPML 1003 RASQSIS 1098 AAS 1175 CQQS 1372 4S 6DHY YDGS YW SYLN SLQ YSTPL - QQFY S TF F1 G 0 00 Fzd FTFSD 208 SAISG 599CARVSRGF 948 RASQSIS 1098 AAS 1175 CQQS 1372 4S 6 YYMS SGFTY AFDYW SYLNSLQ YSTPL - YA S TF G 10 00 Fzd GTFSS 295 GRIIPI 529 CARESVNN 813RASQSIS 1098 AAS 1175 CQQS 1372 4S 6 YAIS LGIAN YYYMDVW SYLN SLQ YSTPL -YA S TF A1 1 00 Fzd FTFSS 227 ALTSY 457 CAKTGRGY 726 RASQSIS 1098 AAS1175 CQQS 1372 4S 6 YAM DGSK AFDIW SYLN SLQ YSTPL - H KFYA S TF C1 1 00Fzd FTFSS 232 KANG 535 CAKAGQQ 696 RASQSIS 1098 AAS 1175 CQQS 1372 4S 6YNM GTTD LDW SYLN SLQ YSTPL - N YA S TF E1 1 00 Fzd FTFTS 249 GGIIPI 499CATVQTNY 977 RASQSIS 1094 AAS 1175 CQQY 1426 4S 6 SAM FGTA YDSSGRFS RWLASLQ VSYPL - Q NYA YRAHYFDY S TF H1 W 1 00 Fzd YTFTN 383 GRIN 537CARGQGYS 855 RASQSIS 1098 AAS 1175 CQQS 1372 4S 6 NFMH PNSG SGWYRGD SYLNSLQ YSTPL - GTNY AFDIW S TF A1 A 2 00 Fzd FAFD 165 KAYG 490 CAKDRGYS 708RASQSIS 1098 AAS 1175 CQQS 1372 4S 6 DYA GTTE SGWYLDY SYLN SLQ YSTPL -MH YA W S TF D1 2 00 Fzd FNFSS 173 SVIYG 653 CARGGSG 836 RASQSIS 1098AAS 1175 CQQS 1372 5S 7 YTMR GGNT GNLSYW SYLN SLQ YSTPL - NYA S TF H0 100 Fzd GTFSS 295 GMIIP 521 CTRPYDAF 1016 RASQSIS 1098 AAS 1175 CQQS 13725S 7 YAIS FLGIT DIW SYLN SLQ YSTPL - NYA S TF A0 2 00 Fzd YTFAS 373 GWIN558 CARLSVWK 890 RASQSIS 1098 AAS 1175 CQQS 1372 5S 8 YGM AGNG WEQVTNSYLN SLQ YSTPL - H NTTY WFDPW S TF C0 A 2 00 Fzd GTFTS 305 GWIN 557CTTGLFPYY 1022 RASQSIS 1098 AAS 1175 CQQS 1372 5S 8 YAIS AGNG RYNWNNDSYLN SLQ YSTPL - NTKY AFDIW S TF E0 S 2 00 Fzd GTFSS 295 GWM 583 CAKWHIG729 RASQSIS 1098 AAS 1175 CQQS 1372 5S 8 YAIS NPNS ATGNWFD SYLN SLQYSTPL - GNTG PW S TF A0 YA 3 00 Fzd YTFTN 383 GGIFP 494 CARDRPTS 792RASQGIS 1072 DAS 1193 CQQSF 1342 5S 8 NFMH IYGIS SWYAFDY NNLN TLQSAPITF - TYA T H0 3 00 Fzd FSFSS 181 SYISS 670 CARYGDYG 954 RASQSIS 1098AAS 1175 CQQS 1372 5S 8 TAMS SGSIT DYW SYLN SLQ YSTPL - HYA S TF F0 4 00Fzd YTFTN 383 GWIN 558 CARVATGN 932 RASQSIS 1098 AAS 1175 CQQS 1372 5S 8NFMH AGNG AFDIW SYLN SLQ YSTPL - NTTY S TF H0 A 4 00 Fzd FTFSS 239 AGISG444 CARGGLLF 831 QASQDIS 1046 KAS 1226 CQQS 1373 5S 8 YWM SGKT DYW NYLNSLES YSTPR - H TFYA TF B0 5 00 Fzd FTFTS 251 GWM 583 CARRTAVA 914RASQSIS 1098 AAS 1175 CQQS 1372 5S 8 SAVQ NPNS GTIDYW SYLN SLQ YSTPL -GNTG S TF F0 YA 5 00 Fzd GTFSS 295 GWIS 573 CARGGWT 841 RASQSIS 1098 AAS1175 CQQS 1372 5S 8 YAIS PYNG NYGGNLDY SYLN SLQ YSTPL - NTNY W S TF G A05 00 Fzd YTFTS 392 GRIN 537 CARVPDF 943 RASQGIS 1075 AAS 1175 CQQT 13925S 8 YYMH PNSG WSGYLDY RTLZ SLQ YSMPI - GTNY W S TF H0 A 5 00 Fzd YTFTY394 GGIIPI 499 CARDSYPY 800 RASQSIS 1098 AAS 1175 CQQS 1372 5S 8 RYLHFGTA GMDVW SYLN SLQ YSTPL - NYA S TF D0 6 00 Fzd GTFSS 295 GRVIP 545CAREYLGS 815 RASQSV 1108 GAS 1213 CQQY 1409 5S 8 YAIS ILGVT FDIW GSNLASRA GSSPP - NYA T FTF F0 6 00 Fzd FTFTG 247 GGILP 509 CARGARLY 822RASQSVS 1114 GAS 1217 CQQR 1335 5S 9 SAVQ IYGTT GFDYW RNLA TRA SNWPI -KYA T TF A0 7 00 Fzd FTFTS 251 GWM 583 CARGRGQ 857 RASQGIS 1076 GAS 1215CLQDY 1272 5S 9 SAVQ NPNS QWLTGYY SALA TLQ NYPFT - GNTG GMDVW S F B0 YA7 00 Fzd FTFSS 233 SYIEN 654 CARAPYYY 748 RASQSIS 1098 AAS 1175 CQQS1372 5S 9 YSMN DGSIT GSGSLFRL SYLN SLQ YSTPL - TYA DYW S TF C0 7 00 FzdGTFN 291 GGIIPI 499 CARAGSGY 740 RASQSIN 1090 AAS 1175 CQQT 1388 5S 9SYAIA FGTA YNFDYW RWLA SLQ YNIPIT - NYA S F D0 7 00 Fzd FSFSS 182 AYINS483 CAKTKLPI 727 RASQSIN 1089 AAS 1176 CQQY 1402 5S 9 YGM RGSL W RNYLGSRV DSWP - H MYYA T PTF F0 7 00 Fzd GSFS 274 GGIIPI 499 CATGYYYD 972RASQGIS 1072 AAS 1175 CQHY 1309 5S 9 GYAI FGTA YYFDYW NNLN SLQ YNLPL - NNYA S TF G 07 00 Fzd GTFT 303 GLVD 520 CARTYRIV 931 RASQTIN 1125 KAS1224 CQQA 1318 5S 9 NNF PEDG GATPRYYY NQLA NLE NSFPV - MH ETIYA YGMDVW TTF H0 7 00 Fzd YIFTD 368 GWIN 562 CARGPRDS 853 RASQSIS 1098 AAS 1175CQQS 1372 5S 9 YYMH PNSG GYYPGGAF SYLN SLQ YSTPL - GTIYA DIW S TF B0 800 Fzd FAFSS 166 SAIDG 592 CARDRQLG 794 RASQSIS 1098 AAS 1175 CQQS 13725S 9 HWM SGGS WAHWYFD SYLN SLQ YSTPL - H TYYA LW S TF D0 8 00 Fzd YTFTG379 GWIN 558 CARDRDY 787 QTSQDIN 1053 KAS 1226 CQQS 1366 5S 9 YYMH AGNGW NNLN SLES YSSPP - NTTY TF G A 08 00 Fzd FTFSS 230 SAIGT 595 CALLVGAA730 QASQDIS 1046 AAS 1179 CLQHK 1276 5S 9 YGM GGGT RGISYYYYY NYLN TLQSFPTF - H YYA GMDVW S C0 9 00 Fzd YTFTS 389 GWIN 558 CARDRPYS 793RASQSVS 1117 GAS 1217 CQQR 1339 5S 9 YAM AGNG SGWYYPAF SNQLA TRA YNWP -H NTTY DIW T PSITF D0 A 9 00 Fzd FNLR 175 SRISN 627 CARDADSS 762 RASQSVS1116 DAS 1188 CQQR 1334 5S 9 RYNM SGSLV GYYRYDAF SNLA NRA NNWL - N YYADIW T YTF E0 9 00 Fzd YTFTD 376 GIINP 513 CARHVYGS 878 RASQSIS 1098 AAS1175 CQQS 1372 5S 9 YYMH SGGS GTYNNWF SYLN SLQ YSTPL - TSYA DPW S TF A10 00 Fzd YTFTS 392 GWM 583 CARGGPIH 834 RASQGIS 1072 AAS 1179 CQQT 13855S 9 YYMH SPNS YYYYYYMD NNLN TLQ NLFPY - ANTG VW S TF D1 YA 0 00 FzdGAFS 265 GRIIP 534 CAKGGWR 715 RASQSVS 1116 GAS 1217 CQQY 1421 5S 9TSSIS VLGT SSFDPW SNLA TRA NSWP - ANYA T LTF H1 0 00 Fzd YTFTS 390 GGFD492 CAKAGDW 695 RASQSIS 1094 AAS 1175 CQQT 1386 5S 9 YDIN PEDG GLYGMDVRWLA SLQ NTFPF - ETIYA W S TF B1 1 00 Fzd FTFTG 247 GGILP 509 CARGARLY822 RASQSVS 1113 GAS 1217 CQQR 1335 5S 9 SAVQ IYGTT GFDYW RKLA TRASNWPI - KYA T TF C1 1 00 Fzd YTFTN 383 GWIN 561 CAREANYD 806 RASQSLR1106 GAS 1217 CQQY 1401 5S 9 NFMH PNSG ILTGYIRPD SKLA TRA ANSP - DTKFAFDIW T WTF D1 A 1 00 Fzd GTFSS 295 GWIN 557 CTTTEYSSS 1025 QASQDIS 1046GAS 1215 CQQLS 1331 5S 9 YAIS AGNG PDYYYGM NYLN TLQ RYPSL - NTKY DVW S FE1 S 1 00 Fzd GTFT 304 GGIIPI 499 CARSSDLRI 922 RASQSVS 1116 GAS 1209CQQY 1413 5S 9 RNSIS FGTA FDYW SNLA NRP GSSPY - NYA T TF G 11 00 FzdYTFAS 372 GWIN 558 CARDGIW 769 RASQSIS 1098 AAS 1175 CQQS 1372 5S 10YDIH AGNG DIFDYW SYLN SLQ YSTPL - NTTY S TF H1 A 1 00 Fzd YIFTD 368GVIFP 551 CARGGSTG 839 RASQSV 1107 AAS 1175 CQQA 1321 5S 10 YYMH VYPTYYGMDVW GRWMA SLQ NTFPF - PDYA S TF E12 00 Fzd GTFSS 295 GRIVP 541CARDTCSS 801 RASQSIS 1098 AAS 1175 CQQS 1372 5S 10 YAIS IVDV TSCSPDYWSYLN SLQ YSTPL - VKYA S TF F1 2 00 Fzd FTFSS 233 SAIGT 595 CAREGWF 810RASQSIS 1098 AAS 1175 CQQS 1372 6S 10 YSMN GGGT GESPFGM SYLN SLQ YSTPL -YYA DVW S TF A0 1 00 Fzd YTFTR 385 GWIS 576 CASPTGMT 959 RASQSIS 1098AAS 1175 CQQS 1372 6S 10 YAVH TFND TNFDYW SYLN SLQ YSTPL - NTNY S TF F0A 1 00 Fzd YIFTD 368 GGIIPI 499 CAKGSYYY 723 RASQGIS 1072 AAS 1166 CQQT1387 6S 10 YYMH FGTA DSSGYYW NNLN NLE SSTPL - NYA DAFDIW T TF H0 1 00Fzd YIFTD 368 GGIIP 507 CARDITGA 775 RASQSIS 1098 AAS 1175 CQQS 1372 6S10 YYMH LFGTT DGMDVW SYLN SLQ YSTPL - DYA S TF A0 2 00 Fzd GTFSS 295GRIIP 533 CARDVCSG 802 RASQGIS 1072 DAS 1189 CQQT 1390 6S 10 YAIS TVGTGSCSPDV NNLN SLES YNTPR - ANYA W TF D0 2 00 Fzd FTFTS 250 GGIIPI 499CARDGSSG 770 RASQGIS 1072 AAS 1175 CLQH 1277 6S 10 SATQ FGTA WYSPNAFNNLN SLQ NGYPI - NYA DIW S TF E0 2 00 Fzd FTFR 200 SRISP 628 CARSPRW 920RSSQSLL 1138 RVS 1251 CMQG 1288 6S 10 MYG DGRT YDAFDIW HSNGYN SRF THWP -MH TTYA YLD S PTF H0 2 00 Fzd YIFTD 368 GWIN 558 CARDPIMF 784 RASESVS1055 GAS 1213 CQQY 1419 6S 10 YYMH AGNG GDQPGWF SNLA SRA NKSPS - NTTYDPW T F A0 A 3 00 Fzd GTFSS 295 GWIN 556 CAREGYDF 811 RASQTIS 1126 EVS1203 CQQS 1378 6S 10 YAIS AGNG WSGPYAF RYLN SLQ YSTP - NTKY DIW G WTF B0A 3 00 Fzd GTFSS 293 GGIIPI 499 CARGGYYY 843 RASQSIS 1098 AAS 1175 CQQS1372 6S 10 NVIS FGTA GMDVW SYLN SLQ YSTPL - NYA S TF C0 3 01 Fzd YIFTD368 GGIIPI 499 CARMSSDY 895 RASQGIS 1072 GAS 1215 CQQA 1312 4S 1 YYMHFGTA YDSSGYYR NNLN TLQ DSFPP - NYA RGMDVW S TF B0 1 01 Fzd GTFSS 295GWIN 558 CARHYYGS 880 RASQSIS 1096 AAS 1175 CQQS 1372 4S 4 YAIS AGNGGSYPDW SHZN SLQ YSTPL - NTTY S TF D0 A 1 01 Fzd GTFSS 295 GWIN 558CARHYYGS 880 RASQSIZ 1105 AAS 1175 CQQS 1372 4S 4 YAIS AGNG GSYPDW ZYZNSLQ YSTPL - NTTY S TF E0 A 1 01 Fzd GTFSS 295 GWM 581 CARHYYGS 879RASQSIS 1098 AAS 1175 CQQS 1368 4S 4 YAIS NPNN GNYRDW SYLN SLQ YSTPF -GNTT S TF G YA 01 01 Fzd FTFSS 223 SGISG 608 CAKPGIAA 725 RASQGIS 1076GAS 1218 CQQS 1373 4S 4 NAM SGGS AGTNNWF SALA TVE YSTPR - H TYYA DPW STF A0 2 01 Fzd FTFSS 227 SGISG 611 CARPSTTSF 902 RASQSVS 1116 GAS 1217CQQY 1403 4S 4 YAM SGSST GMDVW SNLA TRA DTPLR - H YYA T TF B0 2 01 FzdYTFTS 392 PNSG 537 CARVPDF 943 RASQGIS 1076 AAS 1175 CQQT 1392 4S 5 YYMHGTNY WSGYLDY SALA SLQ YSMPI - A W S TF C0 2 01 Fzd GTFST 299 GIINP 513CARAKGSG 744 RASQSIS 1098 AAS 1175 CQQS 1372 4S 5 YAIS SGGS WYVGSAF SYLNSLQ YSTPL - TSYA DIW S TF D0 2 01 Fzd FTFSD 206 GFIRS 490 CARATQEL 758RASQSIS 1098 AAS 1175 CQQS 1372 4S 5 SYMS KAYG LLPYGMDV SYLN SLQ YSTPL -GTTE W S TF E0 YA 2 01 Fzd YTFTS 392 GRIN 537 CARVPDF 943 RASQGV 1079AAS 1175 CQQT 1392 4S 5 YYMH PNSG WSGYLDY STZLS SLQ YSMPI - GTNY W S TFF0 A 2 01 Fzd YTFTS 392 GIISP 516 CARWGDY 951 RASQGIS 1076 ATS 1183 CQQV1399 4S 6 YYMN SGGS GDLYYFDY SALA TLQ NSYPP - TSYA W S TF G 02 01 FzdYIFTD 368 GRIN 537 CARARSSG 751 RASQSVS 1120 AAS 1180 CQQS 1376 4S 6YYMH PNSG WTDAFDI SWLA TLQ YSTPT - GTNY W T F H0 A 2 01 Fzd GTFSS 295GWIN 558 CARHYYGS 880 RASQSIS 1098 AAS 1175 CQQS 1372 4S 6 YAIS AGNGGSYPDW SYLN SLQ YSTPL - NTTY S TF A0 A 3 01 Fzd FTFSS 232 GRIKS 535CARAGDSP 739 RASQSIS 1098 AAS 1175 CQQS 1372 4S 6 YNM KANG DYW SYLN SLQYSTPL - N GTTD S TF B0 TA 3 01 Fzd GTFSS 295 GWIS 574 CARAMWS 745RASQSIS 1098 AAS 1175 CQQS 1372 4S 8 YAIS PYNG YGQQNAF SYLN SLQ YSTPL -YTKY DIW S TF E0 A 3 01 Fzd FTFTS 251 GWM 583 CARRTAVA 914 RASQSIS 1098AAS 1175 CQQS 1372 4S 8 SAVQ NPNS GTIDYW SYLN SLQ YSTPL - GNTG S TF G YA03 01 Fzd YTFTS 387 GRIN 537 CARVKWEL 940 RASQSIS 1098 AAS 1175 CQQS1372 4S 8 SAIH PNSG AIDYW SYLN SLQ YSTPL - GTNY S TF H0 A 3 01 Fzd YIFTD368 GWM 583 CARGGSRY 837 RASQGIS 1074 AAS 1175 CQQS 1368 4S 8 YYMH NPNSDFWSGHW NYLA SLQ YSTPF - GNTG YFDLW S TF B0 YA 4 01 Fzd YTFTG 379 GRIN537 CARDVPKL 804 RASQSIS 1098 AAS 1175 CQQS 1372 4S 8 YYMH PNSG VTRGVAYGSYLN SLQ YSTPL - GTNY MDVW S TF E0 A 4 01 Fzd YSFTT 371 GWIN 558CARAAAGS 736 RASQGIS 1072 EAS 1197 CQQS 1381 4S 8 YGM AGNG YGGGYW NNLNSVA YTSTP - N NTTY S LNSF F0 A 4 01 Fzd FTFSS 231 SAISG 600 CARDLTPF 780RASQSVS 1112 GAS 1216 CQQY 1423 4S 8 YGMS SGGS TQQQLVLG GYLA TRA NYWP -TYYA LL A PAF G 04 01 Fzd FTFTS 251 GRIVP 540 CARSGYNR 919 RASQSIS 1098AAS 1175 CQQS 1372 4S 8 SAVQ AIGFT RGYFDYW SYLN SLQ YSTPL - QYA S TF H04 01 Fzd GTFSS 295 GGIIPI 499 CARVTLGA 949 RASQGIS 1072 DAS 1189 CLQH1278 4S 8 YAIS FGTA SVDAFDIW NNLN SLES NSLPF - NYA TF A0 5 01 Fzd GTFSS295 GWVS 587 CTTDRRYS 1021 RASQSVS 1116 GVS 1223 CQQY 1418 4S 8 YAISPNTG TYFDLW SNLA NRA NIWP - NTVY T RTF B0 A 5 01 Fzd YTFAS 373 GWIN 558CARLSVWK 890 RASQSIS 1098 AAS 1175 CQQS 1372 4S 8 YGM AGNG WEQVTN SYLNSLQ YSTPL - H NTTY WFDPW S TF C0 A 5 01 Fzd GTFTS 305 GWIN 557 CTTGLFPYY1022 RASQSIS 1098 AAS 1175 CQQS 1372 4S 8 YAIS AGNG RYNWNND SYLN SLQYSTPL - NTKY AFDIW S TF D0 S 5 01 Fzd FTFTG 247 GGILP 509 CARGARLY 821RASQZVS 1127 GAS 1217 CQQR 1335 4S 9 SAVQ IYGTT GCDYW RZZA TRA SNWPI -KYA T TF F0 5 01 Fzd FTFSS 226 SAIGT 595 CARKVKGY 884 RVSQGIS 1151 AAS1175 CQQTF 1384 4S 9 SWM GGGT CSGGSCYG SALA SLQ SVPW - H YYA YW S TF G05 01 Fzd FTFSN 216 STISG 648 CARHGRIA 876 RASQSIS 1098 AAS 1175 CQQS1372 4S 9 YAMT SGVS ADIW SYLN SLQ YSTPL - TFYA S TF H0 5 01 Fzd FTFZZ254 GGILP 509 CARGARLY 822 RASQSVS 1114 GAS 1217 CQQR 1335 4S 9 SZVQIYGTT GFDYW RNLA TRA SNWPI - KYA T TF A0 6 01 Fzd FTFSS 233 SYIEN 654CARAPYYY 748 RASQSIS 1098 AAS 1175 CQQS 1372 4S 9 YSMN DGSIT GSGSLFRLSYLN SLQ YSTPL - TYA DYW S TF B0 6 01 Fzd FTFTG 247 GGILP 509 CARGARLY822 RASQSVS 1114 GAS 1217 CQQR 1335 4S 10 SAVQ IYGTT GFDYW RNLA TRASNWPI - KYA T TF C0 6 01 Fzd FTFSR 219 SGIGV 605 CARDAYN 763 RASQSIS1095 AAS 1175 CQQR 1340 4S 10 YAM GGGT WFDPR RYLN SLQ YSTPL - H YYA S TFD0 6 01 Fzd YIFTD 368 GVIFP 551 CARGGSTG 839 RASQSV 1107 AAS 1175 CQQA1321 4S 10 YYMH VYPT YYGMDVW GRWMA SLQ NTFPF - PDYA S TF F0 6 01 FzdFTFSS 227 SAIGA 593 CARDAYN 764 RASQSIS 1098 AAS 1175 CQQS 1372 4S 10YAM GGGT WFDPW SYLN SLQ YSTPL - H YYA S TF G 06 01 Fzd FTFSS 229 SAIGT595 CARDAYN 764 RASQSIS 1098 AAS 1175 CQQS 1372 4S 10 YDM GGGT WFDPWSYLN SLQ YSTPL - N YYA S TF H0 6 01 Fzd FTFSN 210 SAIGT 595 CAREGSYY 809RASQNIG 1080 GAS 1208 CQQY 1417 4S 10 AQM GGGT DWYFDLW SRLA NRA NHWP - SYYA S PLFTF A0 7 01 Fzd IIFSP 313 ALISS 450 CHFGVASV 980 7S 8 NDM GGSTGLNYW - G SYA E0 8 01 Fzd RTFSS 346 AAVS 432 CNLAQRGE 998 7S 8 FVMG ASGGTYW - YTWY H0 A 8 01 Fzd LAFN 317 AAIS 414 CAAGFPTV 680 7S 8 GYTM WSDFVVDGEYD - G NTYY YW A0 A 9 01 Fzd FTLDY 255 ADITS 437 CNAVTYN 994 7S 8YAIS GGST GYTIW - NYA B0 9 01 Fzd LTFSD 319 ASST 464 CNAVTYN 994 7S 8YTVG GGGV GYTIW - FENY C0 A 9 01 Fzd RIFSS 330 PRIPS 590 CEVHNFGA 979 8S4 YAQA DSTTF TYW - YA D0 6 01 Fzd RTFS 344 AVISR 472 CNAVSTD 992 8S 4NYV SGGN WTTDYW - MG TYYT E0 6 01 Fzd RTFST 349 AAIS 414 CNSFPLRL 10028S 4 YGM WSD HDW - G NTYY F0 A 6 01 Fzd LAIDD 318 SYIST 673 CNAVTYN 9938S 5 YYMV SDGS GYSIW - TYYA G 06 01 Fzd LAFN 317 AQIS 460 CNADYGT 985 8S5 GYTM WTG WYGIGW - G GSTD H0 YA 6 01 Fzd LAFN 317 AAIS 412 CNMGLGY 99985 5 GYTM WMS SEYRPLGY - G NTYY W A0 A 7 01 Fzd SAFS 355 AAIT 426CNAVWKF 995 8S 5 NYA WSGA GTTHW - MG RTYY B0 A 7 01 Fzd LTIDD 323 SYISA656 CNAVTYN 994 8S 7 YYVV GDGF GYTIW - TYYA C0 7 01 Fzd GSFS 277 GEIN489 CARDLRFY 778 RSSRSLL 1147 LGS 1237 CMQG 1290 7S 4 GYYW HSGS SSSWRRVGHTSGYNY NRA TRWP - S TNYN MDVW LD S TF F0 9 01 Fzd YTITT 396 GWIN 555CARGWTTI 872 RSSRSLL 1146 LGS 1237 CMQA 1281 7S 4 YAIH ADTG SSLGVWHTNGYN NRA LQTPL - DTAY YLD S TF G S 09 01 Fzd NIFRI 326 AALT 427CNTVTYNA 1004 7S 5 YAIA GQRT GCYKKYW - TNYA H0 ASIT 9 01 Fzd LAFN 317WNG 462 CNARLDAV 988 7S 5 GYTM RYTYY YGHSRYDS - G A W A1 0 01 Fzd NFFS325 GAISR 484 CAAGVTGS 684 7S 5 NYPL TGSG WRYW - G TFYA B1 0 01 FzdRSFSN 335 AVSW 479 CNAVTYN 994 7S 8 YRVA SVGM GYTIW - TYYA C1 0 01 FzdGTFG 288 GLISR 518 CNAVNGRL 991 7S 8 SYAV NAGN NYW - G TLYA D1 0 01 FzdRTFSS 348 AAVS 431 CAAPQSPN 687 7S 8 YSLA ASGA MYIRTDQL - NTYY WWYKYW E1A 0 01 Fzd RSFST 336 TVISG 675 CAAGPTLP 683 8S 1 YPMG SGGS FRYW - TYYAD0 7 01 Fzd RAFS 328 AAIN 406 CNARLSFA 989 8S 1 NYA WSG GGMGYW - MG DSAYE0 YA 7 01 Fzd IKSMF 314 AFITR 438 CNAVSTD 992 8S 1 DMNF GGTT WTRDYW -MG RYG F0 7 01 Fzd LTIDD 322 SYIGT 655 CNAVTYN 994 8S 1 YYMV SDGTGYTIW - TYYA G 07 01 Fzd RVFSS 354 AGIAS 439 CKVHNFGA 983 8S 4 YAQADSTTF TYW - YA H0 7 01 Fzd RIFSS 330 ASIPS 461 CKVHNFEA 982 8S 4 YAQADGTT TYW - FYA A0 8 01 Fzd LTFST 321 AAIN 407 CNSFPLRL 1002 8S 4 YGMWSGR HDW - G STVY B0 A 8 01 Fzd RTLSS 351 ALISL 449 CNAVSTD 992 8S 4YVVG SGAS WTTDYW - TYYA C0 8 01 Fzd IKSMF 314 AFITR 438 CNAVSTD 992 8S 5DMNF GGTT WTRDYW - MG RYG D0 8 01 Fzd RTDG 338 GAIT 486 CNVLAQN 1007 8S5 MQA WSLG DGDYRTYG - MG SAFY E0 A 8 01 Fzd RTFSS 346 AAVS 432 CNAVWKF995 8S 5 FVMG ASGG GTTHW - YTWY F0 A 8 01 Fzd RTFSS 346 AAVS 432CNAVCKFG 990 8S 5 FVMG ASGG TTHW - YTWY G A 08 01 Fzd RTFSS 346 AAVT 434CNAVWKF 995 8S 5 FVMG ASGG GTTHW - YAWY H0 A 8 01 Fzd ITFSF 316 AVFIA470 CNGVTYN 997 8S 8 NSVG GYGA GYTIW - YYA A0 9 01 Fzd HDFS 310 ATIS 468CAAQKPYY 688 8S 8 STYG WGG NGHFYAD - VG TNIA DKHYDHW B0 9 01 Fzd ITFGF315 AVFN 471 CNAVTYN 994 8S 8 DSVG AGYR GYTIW - AYYA C0 9 01 Fzd RTFS350 AAVS 433 CNAVTYN 994 8S 8 WYS WSGV GYTIW - MG STYYP D0 9 01 FzdITFSF 316 AVFIA 470 CIGVTYNG 981 8S 8 NSVG GYGA YTIG - YYA E0 9 01 FzdRTDG 338 GAIT 485 CNVLAQN 1008 8S 8 MQA WSLG DGDYRTY - MG IAFYA W F0 901 Fzd HDFS 311 AAIS 413 CAAQKPYY 688 8S 8 STYG WRGT NGHFYAD - VG NIADKHYDHW G 09 02 Fzd DSVS 160 GRAY 524 CVRDLRPS 1029 RASQSIG 1087 YAS1260 CHQS 1268 1S 8 SNSA YKSR GDLNFDY SSLH QSV GRVP - AWN WYYD W S VTFA0 YA 1 02 Fzd GSISS 283 GSIYH 547 CARFYYDIL 817 RSSRSLL 1142 TLS 1259CMQSI 1295 1S 1 GGYS SGSTY NGYSYFDY DTDDGN HRA QLPW - WS YN W TYLD S TFC0 1 02 Fzd FTFSS 230 AVISY 475 CAKGSVFG 721 RSSQSLV 1140 KISN 1230 CMQA1283 1S 1 YGM DGSN LKAGGYAD HSDGNT RFS TQFPH - H KYYA YW YLS TF D0 1 02Fzd YTFTS 391 GWIS 570 CARDGTPF 772 QGDSLRT 1052 GKN 1219 CNSRD 1300 1S8 YGIS AYNG YSGSYYGS YYAS NRP NSGK - NTNY W S HKVF E0 A 2 02 Fzd DSVS162 GRTY 544 PRLDYW 1034 RSSQSLL 1133 MLS 1240 CMQR 1294 1S 8 SNSG YRSKYDSDDGN SRA LEFPY - AWN YNGY TYLD P TF G A 02 02 Fzd DSVS 160 GRTY 542CARSQATG 921 RSSQNIF 1131 SAS 1254 CQQS 1349 1S 8 SNSA YRSK ERFDYW QSLNSLQ YNSPI - AWN WYN S TF A0 DYA 3 02 Fzd FTFSS 228 SVIST 652 CADGSGTS690 RASQSIS 1098 AAS 1175 CQQS 1372 2S 4 YAMS SGGT HR SYLN SLQ YSTPL -VLYT S TF H0 6 02 Fzd YIFTD 368 GGIFP 493 CAKGSYYY 722 RASQGIS 1071 AAS1166 CQQT 1391 2S 10 YYMH IFGTA DNSGYYW NNIN NLE YSIPFT - NYA DAFDIW T FA1 1 O GFTFS 270 VISGD 677 NFIKYVFA 1033 SGDKLGK 1152 EKD 1200 SSFAG1435 M HYTLS GSYT N KYAS or or NRP or NSLE or P- YYAD SGDNIGS 1153 SG1201 or 1436 18 SVKG FYVH or QSYA R5 DKS NTLSL NRP SG 02 Fzd FTFSS 228SAISG 600 CAKGLWG 718 RASQSVS 1118 GAS 1212 CQQR 1337 7S 5 YAMS SGGSPLLNW SNYLS SRA TNWP - TYYA P PRVTF H0 2 02 Fzd DSVS 161 GRTY 543CTRGNWN 1014 SGTSSNI 1157 GNN 1219 CSAW 1432 7S 8 SNSA YRSK VGLANWGAGYDV NRP DDNL - TWN WYSD H S NGVV B0 YA F 3 02 Fzd RSFSI 334 AAIS 418CNVITIVRG 1006 7S 5 YNTA WSG MGPRAYW - GSTY E0 YA 1 00 Fzd LTFSI 320SAISG 597 CARGVYPY 870 QASQDIS 1046 AAS 1175 CQQS 1371 4S 5 YAM DGALSSKHKPSY NYLN SLQ YSTPL - H TYYA YYYGMDV S FTF D0 W 5 00 Fzd YDFTT 367GGVI 511 CARGYYYG 874 RASQSIS 1098 AAS 1175 CQQS 1372 4S 5 YGIH PAFGMDVW SYLN SLQ YSTPL - ATDY S TF D0 S 4 00 Fzd GTFSS 295 GWIN 558CASGLGYF 957 RASQSIS 1098 AAS 1175 CQQS 1372 4S 5 YAIS AGNG DYW SYLN SLQYSTPL - NTTY S TF B0 A 5 00 Fzd YTFTN 383 GGIIPI 502 CARTLTTPP 929RASQSIS 1098 AAS 1175 CQQS 1372 4S 5 NFMH FGTP YYYGMDV SYLN SLQ YSTPL -HYA W S TF G 03 00 Fzd FTFSN 214 SAIGT 594 CTRDLYGG 1013 KSSQSLL 1036LGS 1237 CMQG 1286 4S 5 SDM GGDT YRDYW HSDGYTY NRA LQTP - N YYA LY S WTFF0 3 00 Fzd YIFTG 369 GRIN 537 CARGGEYS 827 RATQTIS 1129 AAS 1171 CQQY1427 4S 5 YYMH PNSG SGWTYYYY TYLN RLQ YSYP - GTNY YGMDVW S WTS C0 A 4 00Fzd YTFTY 394 GMIN 522 CARDVMD 803 RASQGIS 1072 AAS 1165 CQHL 1303 4S 5RYLH PIGGS VW NNLN ALQ NNFPL - INYA S TF B0 6 00 Fzd FSVG 186 SSISS 634CARGPKT 851 RASQSIS 1098 AAS 1175 CQQS 1372 4S 5 SNYM GNSYI MWEDRPD SYLNSLQ YSTPL - T YYA YW S TF F0 6 00 Fzd FTFST 245 GFIRS 491 CARLTGGA 892RASQGIS 1072 GAS 1211 CQQS 1346 4S 5 YSMI KDYG VAGTHRDY NNLN SLQ HSSPR -GTTE W S TF A0 YA 4 00 Fzd FTFSS 237 SAIGT 595 CARGSSGY 862 RASQSIS 1098AAS 1175 CQQS 1372 4S 5 YVMS GGGT YVAW SYLN SLQ YSTPL - YYA S TF A0 5 00Fzd FTFSN 212 AGVSI 447 CARDQND 785 RSSQSLL 1135 LGS 1235 CMQG 1285 4S 5HYMS DANK SWYRSDY HSDGYTY HRA LQTPH - KYYA W LY S TF F0 5 00 Fzd GTFSS295 GRIN 537 CARGSGYD 861 RASQSIS 1092 AAS 1175 CQQS 1350 3S 1 YAIS PNSGFFDYGMD NNLN SLQ YNTPF - GTNY VW S TF C0 A 1 00 Fzd DTFS 164 GLVD 520CAKASTPM 697 RASQSIG 1085 AAS 1179 CQQN 1333 3S 1 NYVL PEDG VQGAPDY SNLDTLQ YATPR - S ETIYA W S TF H0 1 00 Fzd GTFN 289 GGIIPI 499 CATTQGVY 976RASQSIS 1098 AAS 1175 CQQS 1372 3S 1 RYAIT FGTA SSSWYGG SYLN SLQ YSTPL -NYA GRAFDIW S TF H0 2 00 Fzd YTFTY 394 GRIN 537 CWGGSYY 1030 RASQGIS1072 AAS 1175 CQQA 1316 3S 1 RYLH PNSG GDYW NNLN SLQ NSFPI - GTNY S TFH0 A 4 00 Fzd FTFSS 227 SSISW 638 CARGSGIA 860 TSSQSLL 1162 LGS 1237CMQG 1289 3S 2 YAM NSGR ASGSYW HSDGKTY NRA THWP - H VDYA LY S YTF A0 500 Fzd FTFSN 211 STIAG 643 RGRGAPQ 709 KSSQSLL 1036 LGS 1237 CMQS 12963S 2 AWM SGGR PYYYYGM HSDGKTY NRA LQSPL - S TYYS DVW LY S TF B0 5 00 FzdFSFST 184 SRING 624 CARAIVGA 743 KSSQSLL 1036 LGS 1237 CMQN 1293 3S 2YTMS DGSS TGLNRFKA HSDGKTY NRA THWP - TRYA FDIW LY S LTR F0 5 00 FzdSTFTN 363 SAIGT 595 CARDRVTL 796 RSSRSLL 1145 LAS 1231 CIQNT 1270 3S 2AWM GGGT RGGYSYGT HSNGNT RRA HWPL - S YYA DAFDIW YLR S TR G 05 00 FzdFTLST 257 SRINY 626 CARDRDIV 786 KSSQSLL 1036 MG 1239 CMQG 1287 3S 2 YNMDGSA VVPAQRG HSDGKTY SYR THWP - N TTYA EGGFDPW LY AS LTF H0 5 00 FzdFTFSS 228 SAISG 600 CAKGGRD KSSQSLL 1036 LGS 1237 CMQN 1292 3S 2 YAMSSGGS GYKGYFDY 714 HSDGKTY NRA THWP - TYYA W LY S LTL A0 7 00 Fzd FSFRS178 SAIGT 595 CTTTTVTTS 1026 RTSQSVS 1150 DAS 1187 CQQY 1412 3S 2 YSMSGGGT W SNLA NRA GSSPY - YYA S NF C0 7 00 Fzd FSFSS 183 SHISS 619 CARDGGY768 RASQSIS 1098 AAS 1175 CQQS 1372 3S 2 YGMS GGAT W SYLN SLQ YSTPL -IDYA S TF F0 7 00 Fzd FTFSS 239 SYISG 658 CARDNGYC 783 RASQAIS 1060 KAS1228 CQQA 1313 3S 2 YWM DSGY SGGSCYAT SYLA TLD DTFPF - H TNYA YYGMDVR TTF G 07 00 Fzd FTFSS 240 AVISY 476 CARSYYDS 927 KSSQSLL 1036 LGS 1237CMQT 1299 3S 2 ZZMH DGSN SGYPRKDA HSDGKTY NRA LKAPL - RZYA FDIW LY S TFB0 8 00 Fzd ZSVSS 401 SRINS 625 CARARLLG 750 RSSQYLS 1141 GAS 1213 CQQY1411 3S 2 NYMS DGSTI GYYTPDR SAYLA SRA GSSPT - SYA MDVW T F F0 8 00 FzdFTFN 197 ALISS 451 CARDLMV 777 QASQGIS 1049 AAS 1175 CQQS 1367 3S 2 RHALNGDH GRNKLDY NNLN SLQ YSTPA - S KYYT W S FTF H0 8 00 Fzd FTFSS 225 SGISG611 CARGRVW 859 RSSQSLL 1135 LGS 1237 CMQG 1287 3S 2 SNM SGSST SSRDYWHSDGYTY NRA THWP - N YYA LY S LTF A0 9 00 Fzd FNIRR 174 SAIGT 595CARGDSGS 823 RSSESLL 1130 LGS 1237 CTQTV 1433 3S 2 ZNMZ GGGT YRDYWHSDGKTY NRA QFPIT - YYA LY S F B0 9 00 Fzd FTFSS 224 SGISG 612 CARRLIAV912 RASQGIS 1072 SAS 1252 CQQS 1378 3S 2 SAMH SGTTT AGAEFDP NNLN NLQYSTP - YYR W S WTF C0 9 00 Fzd FTFSN 214 KAYG 536 CARQYYFD 909 RASQSIS1098 AAS 1175 CQQS 1372 3S 4 SDM GTTE YW SYLN SLQ YSTPL - N YA S TF F0 900 Fzd FTFSS 221 SVISS 651 CATASGDF 968 RASQSIG 1086 RAS 1248 CQQT 13983S 4 FGMH GGSP DYW SNLN TLES YTTPR - YYA F H0 9 00 Fzd FTFD 191 AIVSY448 CARQTRG 907 RASQGIS 1072 YAS 1262 CQQS 1345 3S 4 DYA DGTY GTTDGWNNLN SLQ HSPPG - MH KYYS S TF A1 0 00 Fzd FTFSS 222 SAISA 596 CARPIVGA900 RASQSIS 1098 AAS 1175 CQQS 1372 3S 4 HSTH SGDS TAFDIW SYLN SLQYSTPL - TFYA S TF B1 0 00 Fzd FTZSS 264 SYSSG 674 CARGVVGS 868 RASQSIV1104 DAS 1186 CQQG 1329 3S 4 YSMN NSGY GAFDIW SYLN NLQ YSAP - TNYA S WTFG 10 00 Fzd FTFSD 208 SAIDG 591 CARAIPGD 742 RASQSIS 1098 AAS 1175 CQQS1372 3S 4 YYMS AGRT YDYW SYLN SLQ YSTPL - YYT S TF B1 1 00 Fzd FTFTS 252GGIIPI 496 CARTGRGY 928 RASQSIG 1085 AAS 1179 CQQS 1373 3S 4 YAM FGIANYGMDVW SNLD TLQ YSTPR - H YA S TF C1 1 00 Fzd FTFSS 234 SYISG 658CARAGVAT 741 RASQSIS 1098 AAS 1175 CQQS 1372 3S 4 YSMS DSGY IAFDYW SYLNSLQ YSTPL - TNYA S TF D1 1 00 Fzd FTFD 192 SAISG 600 CTTPNYYD 1023RASQSIS 1098 AAS 1175 CQQS 1372 3S 4 DYG SGGS SR SYLN SLQ YSTPL - MHTYYA S TF F1 1 00 Fzd GTFSS 295 GWIN 558 CARHYYGS 880 ZPZQTZZ 1164 PAS1242 CQQS 1372 3S 4 YAIS AGNG GSYPDW SHLN SLQ YSTPL - NTTY S TF E1 A 200 Fzd FTFST 244 SYISS 671 CARGGLDG 830 RASQGIS 1072 AAS 1179 CQQG 13264S 4 YGM SSSAI PIDYR NNLN TLQ NNFPF - H YYA S TF A0 1 00 Fzd FTVSS 260SLVSF 621 CARLGSTP 887 RASQGIS 1072 AAS 1175 CQQY 1428 4S 4 HSMG DGSKDYW NNLN SLQ YTYPY - EHYA S TF G 01 00 Fzd FTFSS 230 AVISY 475 CASDPVTA956 RASQSIS 1098 AAS 1175 CQQS 1372 4S 4 YGM DGSN ATR SYLN SLQ YSTPL - HKYYA S TF C0 2 00 Fzd FSFSS 183 SGISG 610 CAKDGYW 699 RASQSIS 1098 AAS1175 CQQS 1372 4S 4 YGMS SGRS SYLN SLQ YSTPL - TYYA S TF D0 2 00 FzdFTFSS 227 WNG 606 CARPAGSA 899 RASQGIS 1072 DAS 1185 CHQSY 1269 4S 4 YAMGSTG QNWFDP NNLN NLE SIPRTF - H YA W T A0 3 00 Fzd FSFSR 180 SGVG 617CARDGSW 771 RASQDV 1066 DAS 1191 CQQG 1328 4S 4 YGMS GSGG DTWLA TLETYNIP - STZYA WTF B0 3 00 Fzd YTFTS 388 GIINP 513 CARQIGWE 906 QASQDIS1047 AAS 1179 CQQAI 1315 4S 4 YAIS SGGS LMPDIW SYLN TLQ SFPLTF - TSYA SC0 3 00 Fzd ZZZTD 403 GGM 510 CANGSYA 732 RASQSIS 1098 AAS 1175 CQQS1372 4S 5 YYZQ NZNR QHLW SYLN SLQ YSTPL - GNTG S TF C0 YA 5 00 Fzd FTFSS239 STISP 649 CAKDKVPY 703 RASQSIS 1098 AAS 1175 CQQS 1372 4S 5 YWMSGLYI SYGPNFDY SYLN SLQ YSTPL - H YQA W S TF G 05 00 Fzd FFFSG 169 QDGS459 CARVFPLH 934 QASQDIS 1046 KAS 1226 CQQA 1319 4S 5 YWM EKYY DYW NYLNSLES NSFPY - S V TF E0 6 00 Fzd FPFST 176 AGIS 446 CARSGPAA 918 RSSQNVS1132 GAS 1217 CQHR 1305 4S 5 FSMN WNS MVYYYYG SYLA TRA ANWP - GTIDY MDVWT QTF C0 A 6 00 Fzd FTLSS 256 SAIGT 595 CAAPDYW 686 RASQSIS 1098 AAS1175 CQQS 1372 4S 6 HHM GGGT SYLN SLQ YSTPL - N YYA S TF E0 7 00 FzdFSFSK 179 SSIDG 629 CARPYYYD 904 QASQDIT 1048 KAS 1229 CQQS 1356 4S 6KYMT NGDH SSGYDPM NYLN TLES YSAPY - VFYA GDYW TF F0 7 00 Fzd FTVSS 261SAIGT 595 CAQGTYW 735 RASQSIS 1098 AAS 1175 CQQS 1372 4S 6 NYM GGGT SYLNSLQ YSTPL - N YYA S TF C0 8 00 Fzd FTFD 193 SAVS 604 CARGGNY 833 RASQSIS1102 EAS 1198 CQQT 1397 4S 6 DYYM GNGG GSGDYW ZWLA TLQ YTPPF - N GTFY STF F0 A 8 00 Fzd GTLN 308 GRIIPI 526 CARDRRGY 795 RASQAIS 1058 DAS 1185CQQA 1324 4S 6 NHTL FGTA GMDVW NSLA NLE YSFP - S NYA T WTF G 09 00 FzdFTFSD 208 SGIN 607 CARIGAGG 881 RASQSIS 1098 AAS 1175 CQQS 1372 4S 6YYMS WNS AFDIW SYLN SLQ YSTPL - AKIGY S TF B1 V 0 00 Fzd FIFSD 170 AVITS477 CARNGIAA 896 RASQSIS 1101 GAS 1210 CQQS 1362 4S 6 YYMS GGTF AEDYWTYLS SLES YSPPF - KYYA TF H1 0 00 Fzd FTFSS 226 SGIS 615 CARYSSGG 955RASQSIS 1098 AAS 1175 CQQS 1372 4S 6 SWM WNS SLDYW SYLN SLQ YSTPL - HGSIGY S TF B1 A 1 00 Fzd YZFZZ 400 GRIN 537 CARARSSG 751 RASQSVS 1120AAS 1180 CQQS 1376 4S 6 ZYMH PNSG WTDAFDI SWLA TLQ YSTPT - GTNY W T F D1A 1 00 Fzd FTFSS 228 SSISG 632 CARPYSSS 903 RASQSIS 1098 AAS 1175 CQQS1372 4S 6 YAMS GGRH RQGDYW SYLN SLQ YSTPL - TYYA S TF F1 1 00 Fzd YIFTD368 GWIN 564 CARDRPGF 790 RASQSVS 1121 GAS 1213 CQQY 1400 4S 6 YYMH PNSGDPW SYLA SRA AISYTF - GTNY T G A 11 00 Fzd FTFSS 238 SYISG 658 CAKGIRWF716 RASQSIS 1098 AAS 1175 CQQS 1372 4S 6 YWIH DSGY DPW SYLN SLQ YSTPL -TNYA S TF B1 GWM 2 00 Fzd YIFTD 368 NPNS 583 CASSHYAP 962 RASQGIS 1076RTS 1250 CQQS 1378 4S 6 YYMH GNTG GMDVW SYLA TLES YSTP - YA WTF C1 2 00Fzd FTVG 258 SSITT 640 CARGKEGR 844 RASQSIS 1098 AAS 1180 CQQS 1358 4S 7NNY TSTLY YSNYEAA SYLN TLQ YSIPFT - MS A W T F F1 2 00 Fzd FTFRS 202SLISG 620 CARREPLY 910 RASQSIS 1098 AAS 1175 CQQS 1372 5S 7 YGM SGDNSSRRGAFDI SYLN SLQ YSTPL - H TNYA W S TF B0 1 00 Fzd FTFSS 234 SAISG 600CTRTIVGAT 1017 RASQGIS 1072 KAS 1227 CQQS 1360 5S 7 YSMS SGGS PHYW NNLNSLQ YSLPY - TYYA S TF C0 1 00 Fzd FTVSS 262 SAISG 598 CAKGAGY 711RASQSVS 1119 GAS 1213 CQQR 1338 5S 7 NYMS SGAT GSGSWQA SSYLS SRA YKSYT -TTYA AW T F F0 1 00 Fzd YSFTN 370 GRIIPI 527 CARGTFLE 866 RASQSIS 1098AAS 1175 CQQS 1372 5S 8 YAM FGTA WLLTNYG SYLN SLQ YSTPL - H ZYA MDVW STF B0 2 00 Fzd GTFSS 297 GWIG 554 CATGWPR 971 RASQSVS 1116 NTS 1241 CQHY1308 5S 8 YVIS PHNG YYYGMDV SNLA NRA NNWP - NTNY W T FTF D0 A 2 00 FzdYTFTS 392 GGIIPI 500 CARLPYYD 889 RASQSVS 1122 DAS 1188 CQQR 1336 5S 8YYMH FGTA FWSGYYG TNLA NRA SNWP - ZYA GRTGFDY T PQITF G W 02 00 FzdYTFTY 394 GWIN 558 CARASLYY 753 QASQDIS 1044 AAS 1175 CQQS 1372 5S 8RYLH AGNG DYVWGSY HYLN SLQ YSTPL - NTTY RHYYFDY S TF H0 A W 2 00 FzdGTFSS 295 GIINP 512 CATSFGGG 975 RASQSIN 1091 GAS 1213 CQQY 1413 5S 8YAIS SGGR WIVVDTSL SNLA SRA GSSPY - TTYA WYW T TF B0 3 00 Fzd GSFS 275GGIIPI 499 CRVDAFDI 1009 RASQSVS 1119 DTS 1194 CQQY 1408 5S 8 GYAIS FGTAW SSYLS NRA GSSPI - NYA T TF C0 3 00 Fzd FTFTS 251 GGIIPI 499 CARSSGW923 RTSQSISS 1149 AAS 1181 CQQSF 1343 5S 8 SAVQ FGTA QNRFAFDI YLN TSQSSWTF - NYA W S E0 3 00 Fzd YTFTY 394 GWIN 557 CATDLPVR 970 RASQSIS 1098AAS 1175 CQQS 1372 5S 8 RYLH AGNG KGFTYYDIL SYLN SLQ YSTPL - NTKYTGSYGMD S TF F0 S VW 3 00 Fzd YTFTN 383 GGIIPI 498 CARGLRYF 848 QASHDI1039 AAS 1175 CQQS 1365 5S 8 NFMH FGTA DWPQGIY NIALN SLQ YSSPL - NHAYYYGMDV S TF B0 W 4 00 Fzd YTFTS 392 GRIN 537 CARGGLLF 831 RASQSIS 1098AAS 1175 CQQS 1372 5S 8 YYMH PNSG DYW SYLN SLQ YSTPL - GTNY S TF C0 A 400 Fzd FTFST 246 STIGT 645 CARVGWL 938 RASQSIS 1098 AAS 1175 CQQS 13725S 8 YSMS GGGT RFLDYW SYLN SLQ YSTPL - YYA S TF D0 4 00 Fzd GTFSS 295GWM 585 CARNNFLR 897 RASQSIS 1098 AAS 1175 CQQS 1372 5S 8 YAIS SPSSGAFDIW SYLN SLQ YSTPL - NAGY S TF G A 04 00 Fzd FAFSS 167 SRIDT 622CARAPSYS 747 RASQSIS 1098 AAS 1175 CQQS 1372 5S 8 YAMS DGST SGWYVRW SYLNSLQ YSTPL - TYVA S TF A0 5 00 Fzd YTFTY 395 GIINP 513 CARELLPM 812RASQGIS 1072 RAS 1247 CQQA 1320 5S 8 YAM SGGS TTVTSPFI NNLN SLQ NSYPL -H TSYA W S TF C0 5 00 Fzd GTFSS 295 GGIIPI 499 CAIRAFDI 694 RASQSIS 1098AAS 1175 CQQS 1372 5S 8 YAIS FGTA W SYLN SLQ YSTPL - NYA S TF E0 5 00Fzd ZTFSZ 402 SSISS 635 CARVRSKA 947 RASQSVS 1119 AAS 1172 CQQY 1424 5S8 YDM SSHY VAGTLPKR SSYLS RRA SNWP - H KYYA LFDIW T FTF C0 6 00 FzdYTFTS 392 GWM 583 CARGNPTS 850 QASQDIS 1045 SAS 1253 CQQS 1354 5S 8 YYMHNPNS GHIVVVPA NRLN RLQI YRTPR - GNTG ATFSDYW TF E0 YA 6 00 Fzd GTFSZ 302GWM 579 CARWAFPI 950 RASQSIS 1098 AAS 1175 CQQS 1372 5S 8 ZTIS NPDSPNAFDIW SYLN SLQ YSTPL - GKTG S TF G YA 06 00 Fzd YTFTN 383 GGIFP 494CARDRPSS 791 RASQGIS 1072 DAS 1193 CQQSF 1342 5S 8 NFMH IYGIS SWYAFDYNNLN TLQ SAPITF - TYA W T H0 6 00 Fzd GTFSZ 301 GGIIPI 499 CARGGLLR 832RASQSIS 1097 GAS 1217 CQQY 1407 5S 9 YAIS FGTA FGDGWG SKSLA TRA GIAPT -NYA MGMDVW T F A0 8 00 Fzd YTFTD 375 GWIN 558 CARASSWY 755 RASQSIS 1098AAS 1175 CQQS 1372 5S 9 YHM AGNG LHYYYGM SYLN SLQ YSTPL - H NTTY DVW STF C0 A 8 00 Fzd FIFSZ 171 SSISA 631 CARRGYSS 911 RASQSIS 1098 AAS 1175CQQS 1372 5S 9 YAMS AGAY GWRDAFD SYLN SLQ YSTPL - KYYA IW S TF E0 8 00Fzd YTFTS 392 GWIN 558 CAKDVNY 710 QASQGIS 1050 AAS 1175 CQQT 1395 5S 9YYMH AGNG W NYLN SLQ YSTPT - NTTY S TF F0 A 8 00 Fzd GTFSS 295 GRIIPI531 CARDRLAF 789 RASQSIS 1098 AAS 1175 CQQS 1372 5S 9 YAIS LGTP DYW SYLNSLQ YSTPL - NYA S H0 TF 8 00 Fzd FAFSS 166 SAISV 602 CARWGKR 952 RASQSIG1085 RAS 1249 CQQS 1375 5S 9 HWM SGGT LRGSPYYF SNLD TLQ YSTPS - H TFYADYW S F A0 9 00 Fzd FTFSI 209 SGIS 614 CARGPLPT 852 RASQSIS 1098 AAS1175 CQQS 1372 5S 9 YGM WNS KIGGHYM SYLN SLQ YSTPL - H GNIG DVW S TF F0YA 9 00 Fzd FTFST 241 AVMY 478 CARLSYYY 891 RASQGIS 1072 AAS 1175 CQQG1327 5S 9 XWM SGGT DSSGPKGD NNLN SLQ NNFPL - S TYYA AFDIW S TF B1 0 00Fzd FSLSS 185 SSISS 636 CARSGMV 917 RASQDIG 1061 AAS 1175 CQKY 1310 5S 9YGM SSSYI KWLRSFDY SFLA SLQ NRAPF - H YYA W S TF C1 0 00 Fzd FTFTS 249GVIN 552 CARGYGDY 873 RASQSIS 1098 AAS 1175 CQQS 1372 5S 9 SAM PGSGVWGENYF SYLN SLQ YSTPL - Q GTSY DYW S TF E1 N 0 00 Fzd YTLSN 397 GWIS568 CARFDYFG 816 RASQSIS 1098 AAS 1175 CQQS 1372 5S 9 YGIS AYNG GMDVWSYLN SLQ YSTPL - DTKY S TF F1 A 0 00 Fzd YTFTR 385 GGIIP 495 CAADRSPY679 RASQGIS 1072 QAS 1244 CQQS 1352 5S 9 YAVH FFNT YYDSSGYY NNLN SLDYNVPY - VNYA PDAFDIW S TF G 10 00 Fzd FTFSS 229 SGIS 615 CAKGSLLL 720RASQSIS 1092 DAS 1192 CQQS 1350 5S 9 YDM WNS GYYGMDV NNLN TLK YNTPR - NGYIGY R TF A1 A W 1 00 Fzd FTZSS 263 SSISG 633 CAREAGTT 805 RASQSIS 1098AAS 1175 CQQS 1372 5S 10 YDM LGGS GGWFDP SYLN SLQ YSTPL - H TYZA W S TFB1 2 00 Fzd FTFSD 204 STIGP 644 CARASTSG 756 RASQSVS 1123 GAS 1217 CQQY1406 5S 10 HYM AGDT DYSLW TSYLA TRA GASP - D YYP T WTF D1 2 00 Fzd YTFTN384 GLVC 519 CARRTSAS 915 RASQSIS 1098 AAS 1175 CQQS 1372 6S 10 YCTRPSDG DIW SYLN SLQ YSTPL - STSYA S TF B0 1 00 Fzd FTFTZ 253 GGFD 492CTTDPLELP 1020 RASQGIS 1076 SAS 1252 CQQAI 1315 6S 10 SAVQ PEDG WYW SALANLQ SFPLTF - ETIYA S C0 1 00 Fzd YTFTG 379 GIINP 514 CARDLTYY 781RASQSVT 1124 GAS 1217 CQQY 1416 6S 10 YYMH SSGR YDSSGHSP SSLA TRA NDWP -TDYA LGAFDIW T PTF E0 1 00 Fzd FTFSD 203 AGISG 443 CARDSDF 797 KSSQSVL1037 STN 1257 CQHR 1306 6S 10 FGM GGGS WYYYGMD YSSNNK TRS NFF - N TDYAVW NYLA S G 01 00 Fzd VSFS 366 AYINS 482 CAREEWEL 807 RASQSIS 1098 AAS1175 CQQS 1372 6S 10 GYA GSSE FGMDVW SYLN SLQ YSTPL - MH MNY S TF B0 A 200 Fzd YTVTS 399 GGIIPI 497 CAKGGQW 713 RASQSIS 1098 AAS 1175 CQQS 13726S 10 YAM FGTA LYGMDVW SYLN SLQ YSTPL - H KYA S TF G 02 01 Fzd YTFTS 392GWVS 588 CARDEGA 766 RASQSIS 1098 AAS 1175 CQQS 1372 4S 1 YYMH PSSGGYYYYYM SYLN SLQ YSTPL - NTAY DVW S TF A0 A 1 01 Fzd FTFSN 216 SAIGT 595CATAYRRP 969 RSSQSLL 1134 LGS 1237 CMQN 1293 4S 2 YAMT GGGT GGLDVWHSDGKTY NRA THWP - YYA LY S LTR C0 1 01 Fzd FTFSS 227 SVIST 652 CARGGSSD838 RASQSIS 1098 AAS 1175 CQQS 1372 4S 4 YAM SGDT VR SYLN SLQ YSTPL - HVLYT S TF F0 1 01 Fzd FTFSN 217 SYISS 672 CARAALGY 737 RASQGIS 1072 AAS1171 CQQS 1363 4S 4 YGM SSSTI CTGGVCPP NNLN RLQ YSPPL - H YYA VDYW S TFH0 1 01 Fzd YTFTN 383 GIIZP 517 CAKGDYG 712 RASQSIS 1098 AAS 1175 CQQS1372 4S 7 NFMH GGGR ALDYW SYLN SLQ YSTPL - TIYA S TF C0 3 01 Fzd FNFSS173 SVIYG 653 CARGGSG 836 RASQSIS 1098 AAS 1175 CQQS 1372 4S 7 YTMR GGNTGNLSYW SYLN SLQ YSTPL - NYA S TF D0 3 01 Fzd YTFTN 383 GGIIP 506CARLVVRG 893 RASQSIS 1098 AAS 1175 CQQS 1372 4S 8 NFMH LFGTA GYGMDV SYLNSLQ YSTPL - NYA W S TF F0 3 01 Fzd GTFSS 295 GWIS 575 CARADDYY 738RASQSIS 1098 AAS 1175 CQQS 1372 4S 8 YAIS SFNG DSSGYYYG SYLN SLQ YSTPL -NTKY FDYW S TF A0 A 4 01 Fzd FTFSS 235 SRING 623 CARGWAG 871 RASQSIS1098 AAS 1175 CQQS 1372 4S 8 YTMN DGSN FDYW SYLN SLQ YSTPL - TNYA S TFC0 4 01 Fzd HTFS 312 GWIN 558 CARDLSPM 779 RASQSIS 1098 AAS 1175 CQQS1372 4S 8 GYHI AGNG VRGVISGM SYLN SLQ YSTPL - H NTTY DVW S TF D0 A 4 01Fzd YTFTN 383 GIISP 515 CAKGDYG 712 RASQSIS 1098 AAS 1175 CQQS 1372 4S 8NFMH GGGR ALDYW SYLN SLQ YSTPL - TIYA S TF E0 5 01 Fzd FTFG 195 SSLS 641CARDSSSG 798 RASQSIS 1095 AAS 1175 CLQH 1275 4S  10 NYD WNS WYASYYG RYLNSLQ HSYPF - MN GTIVY MDVW S TF E0 A 6 02 Fzd YTLTT 398 GWM 582 CARGALG819 QASQDIS 1046 AAS 1173 CQESY 1302 7S 5 WYM NPNS MDVW NYLN SLH SSPYT -X GNTA T F C0 YA 2 02 Fzd YTFTG 378 GWM 583 CARGTGGF 867 RASQSIS 1098AAS 1175 CQQS 1379 7S 8 HYM NPNS DYW SYLN SLQ YSTPY - H GNTG S TF E0 TA3 02 Fzd YTFTG 377 GWM 580 CARSTPFD 925 RASHDIG 1057 AAS 1179 CQQS 13557S 8 HYIH NPISG PW TFLA TLQ YRTPY - NTGY S TF F0 A 3 02 Fzd YTFTH 380GWIN 559 CARGDYDF 824 QATQNI 1051 KAS 1229 CQQS 1372 7S 8 SYIH AKSGWSGYHEY KKYLN TLES YSTPL - GTFY YYYGMDV TF G A W 03 02 Fzd YTFTS 392GWIN 564 CARAPLDG 746 RSSQSLL 1138 LGS 1237 CMQA 1282 7S 8 YYMH PNSGSGSYYVD HSNGYN NRA LQTPQ - GTNY W YLD S TF H0 A 3 02 Fzd YTFTN 382 GWIS571 CARDCSGG 765 RASQSIS 1093 AAS 1169 CQQA 1323 7S 8 HFMH PNRG SCYSHFDYRSLA NLQ YSFPQ - GTNY W S TF A0 A 4 02 Fzd FTVG 259 SAIGT 595 CAKDITPY702 RASQAIS 1059 AAS 1175 CQQTF 1383 7S 8 SWY GGGT GDYSILSH NYLN SLQSPPLT - MS YYA W 5 F B0 4 02 Fzd YTFTS 386 GGIIPI 503 CARDSSSW 799RASQGIN 1067 QAS 1243 CQQT 1393 7S 8 HWM FGTT YSYYYYYM NYLA NLE YSSPL -H NYA DVW S TF C0 4 02 Fzd YTFTT 393 GWIY 578 CTTDLRYD 1019 QASQDI 1040AAS 1175 CQQS 1377 7S 8 YFMH PNSG SSGPAAFDI DNYLN SLQ YSTPV - GTKY W STF D0 A 4 02 Fzd FTFSD 205 SGISG 609 CATYGDFG 978 RASQGIR 1069 AAS 1179CQQA 1324 7S 8 HYMS SGGT YFDLW NDLG TLQ YSFP - TYYA S WTF E0 4 02 FzdGSFST 279 GRIIP 532 CVKDRAW 1027 RASQGIR 1068 AAS 1174 CQQS 1359 7S 8SVFG LFGTT GFDYW NDLA SLQ YSKPT - NYA R F C0 5 02 Fzd YTFTS 392 GWIN 560CARGGFVF 828 QASQDIS 1046 ASS 1182 CQQS 1356 7S 8 YYMH PKSG DYW NYLN TLQYSAPY - GTNY T TF D0 A 5 02 Fzd GTFSS 295 GMIN 523 CARQAGLH 905 RASQGIT1077 AAS 1168 CQQY 1422 7S 8 YAIS PSGG CSSTSCYLG KSLA NLQ NTFPI - STTYANWFDPW L TF E0 5 02 Fzd GTFN 290 GGIIP 508 CAKGNWA 719 RASQSIS 1100 GAS1217 CQQY 1411 7S 8 RYGIS RLGA FDIW TYLA TRA GSSPT - TDYA T F F0 5 02Fzd GTFSS 295 GWIS 572 CARGVWT 869 RASQSIS 1098 YAS 1261 CQQS 1368 7S 8YAIS PYNG TPMGGGG SYLN SLQ YSTPF - NTKY NWFDPW N TF G A 05 02 Fzd GTFG286 GWIN 564 CARETTDY 814 RASQSIG 1088 DAS 1185 CQQA 1317 7S 8 NYGI PNSGYYGMDVW TYLN NLE NSFPL - N GTNY T TF H0 A 5 02 Fzd GTFSS 294 GVID 550CARVLPGD 941 RASQGIS 1072 KAS 1226 CQQA 1316 7S 8 YAIN PSTG SSGWYRG NNLNSLES NSFPI - GTNY YYYYYGM TF A0 A DVW 6 02 Fzd GTFTS 306 GWIN 566CARDLDSG 776 RASQGV 1078 DAS 1186 CQQH 1330 7S 8 YPIS TYNG FDLW GDYLANLQ NAYPL - NTIYA S TF C0 6 02 Fzd GTFSS 295 GWIS 569 CARGGYSY 842RASQDIS 1064 KAS 1229 CQQS 1348 7S 8 YAIS AYNG GTVFDYW SWLA TLES YGAPL -HTNY TF D0 A 6 02 Fzd YTFTK 381 GGIIPI 499 CARGLPPA 847 RASQNV 1082 SAS1252 CQQS 1368 7S 8 DYM FGTA AGGGGYF NDWLA NLQ YSTPF - H NYA QHW S TF E06 02 Fzd FTFSS 227 AVTW 480 CAKDLVPY 705 RASQSIS 1098 GAS 1207 CQQS 13727S 8 YAM YDGS CSGGSCPP SYLN NLQ YSTPL - H NKYY SGW S TF F0 A 6 02 FzdYTFTD 376 GWM 584 CARGKSGS 846 RASQSV 1111 GTS 1222 CQQY 1404 7S 5 YYMHSPNS FDYW NNTYVA TRA DTSPP - FA GNAG T TF G 06 02 Fzd YTFTG 377 GIINP513 CARGFCSG 825 RASQSIS 1098 AAS 1175 CQQS 1382 7S 5 HYIH SGGS GSCLWYGSYLN SLQ YTTPF - TSYA MDVW S TF H0 6 02 Fzd GTFG 287 GGIIPI 499 CAKDNGW706 RASQSIS 1099 AAS 1175 CQQS 1379 7S 5 SYAIT FGTA YFDLW TNVN SLQYSTPY - NYA S TF A0 7 02 Fzd GTFSS 295 GRIN 1461 CARATRVS 1472 KSSQSVL1498 STN 1257 CQQY 1544 9S 1 YAIS PHNG AAGTVHF HSSNNK TRS YSTPF - NTNYQHW NYLA S TF B0 A 1 02 Fzd YTFTR 1452 GWM 583 CARVRFLE 1494 RASQSLS1512 DAS 1520 CQQAI 1315 9S 2 YYIH NPNS EMDVW SWLA TLQ SFPLTF - GNTG SD0 YA 1 02 Fzd GTFSS 1457 GIINP 513 CARGDIVA 1478 RASQDIS 1500 GAS 1522CQQA 1318 9S 2 YGIS SGGS TMGMKKV NNLN HLQ NSFPV - TSYA DYYYYMD T TF C0VW 2 02 Fzd YTFTR 1453 GWM 583 CARGIGYW 1481 RASQGIS 1074 AAS 1515 CLQYN1528 9S 2 YYLH NPNS NYLA RLQ TYPW - GNTG T TF F0 YA 2 02 Fzd GTFST 298GDIIPI 1458 CARELGLG 1476 RSSQSLL 1138 LGS 1525 CMQA 1281 9S 2 YAIS FGSAWFDPW HSNGYN SRA LQTPL - NYA YLD S TF H0 2 03 Fzd YTFTD 1449 GWM 1467CARGDINY 1477 RASQSIS 1098 KAS 1523 CQQAI 1315 0S 7 YYMH NPNS GNFDYWSYLN TLH SFPLTF - GSTG N A0 YA 2 03 Fzd YTFTD 1449 GWM 583 CARQGGSY 1488RASQSIT 1511 KTS 1524 CQQG 1531 0S 3 YYMH NPNS SMGLDPW TYLN SLQ DSFPY -GNTG S TF B0 YA 2 02 Fzd YTFTG 379 GWIN 1463 CARSYYGV 1492 RASQSIS 1098AAS 1516 CQQSF 1532 9S 3 YYMH PNSG IDAFDIW SYLN SLQ RLPLT - NTGY T F E0A 3 02 Fzd YTFTN 1451 GWM 583 CAREDDF 1474 RASQSIIS 1509 AAS 1175 CQQS1535 9S 3 YYMH NPNS WSGGGM YLN SLQ WRFP - GNTG DVW S YTF G YA 03 03 FzdFTFSD 1438 SAISG 1468 CAREGLRG 1475 KSSQSVL 1037 WA 1526 CQQY 1545 0S 3YYMS SGHS WSIFDIW YSSNNK STR YSTPP - TYYA NYLA ES TF E0 3 02 Fzd YTFTD1447 GWA 1462 CARSRLRW 1491 RASQTIS 1513 DAS 1185 CQQS 1536 9S 3 HYFHNPSS DWYFDLW SYLN NLE YSIPLT - GNTG T F D0 TA 5 03 Fzd FSFSS 1437 SAIGT595 CANPKHY 1470 RASQGV 1505 AAS 1175 CQQY 1543 0S 3 HAMS GGGT W STYLASLQ YSSPQ - YYA S TF H0 3 02 Fzd YTFSR 1446 GWM 583 CARGGHT 1479 RASQSVS1120 AAS 1175 CQQA 1529 9S 3 HYIH NPNS NHW GYSSGWY SLQ FRFPP - GNTG SWLAS TF B0 YA 6 02 Fzd YWFT 1455 GWM 1465 CARRSSSW 1489 RASQNIN 1506 AAS1175 CQQY 1542 9S 3 ASYM KPDS GWYFDLW SWLA SLQ YSFPL - H GNTG S TF E0 YA6 2 Fzd YTFAK 1444 GWM 583 CARHKRHT 1487 RASQSIS 1098 AAS 1175 CQQS 15339S 3 YYIH NPNS PYAFDIW SYLN SLQ HSTPL - GNTG S TF H0 YA 6 02 Fzd YTFTD1448 GWIS 570 CARGSGYF 1486 RASQSIS 1510 GAS 1215 CQQA 1324 9S 3 SYIHAYNG DLW KWLA TLQ YSFP - NTNY S WTF G A 07 02 Fzd YTFTG 378 GWM 583CARVGDY 1493 RASRTVY 1514 DAS 1519 CQQS 1537 9S 3 HYM NPNS DRFNWYF NFLANLR YSTPP - H GNTG DLW T TF H0 YA 8 02 Fzd GTFSS 1456 GWIS 570 CARANRGL1471 RASQSIA 1507 GAS 1211 CQQS 1351 9S 3 YAIT AYNG RKNYYYG RYLN SLQYNTP - NTNY MDVW S WTF F0 A 9 03 Fzd YTFTS 1454 GIINP 1459 CARWTTV 1495RASQGIR 1502 DAS 1518 CQQSS 1534 0S 3 SYIH SGGG VTGAAFDI NDLN NLGRIPPTF - AVYA W T F0 4 02 Fzd YSFTG 1442 GWIN 564 CARDHGT 1473 RASQGIS1503 AAS 1175 CQQS 1538 9S 3 YYLH PNSG MIAVAGTF KYLA SLQ YSTP - GTNYDYYYYMD S WTF A1 A VW 0 02 Fzd YTFN 1445 GIVN 1460 CARGGNY 1480 QASQDIS1046 GAS 1521 CQQT 1540 9S 3 GYYM PSGG GRWLQPW NYLN ALR KSFPL - H GTNYYFDLW S TF B1 A 1 02 Fzd HTFTS 1440 GWM 1466 CARGLGYF 1483 RASQDIS 1501AAS 1517 CQQA 1324 9S 3 HYM NPNS DLW RGLG TLY YSFP - H ANAG R WTF D1 YA1 03 Fzd YSFTN 1443 GWM 583 CARSPDF 1490 RASQSIG 1508 AAS 1175 CQQA 15300S 7 YYMH NPNS WSGEGYF NYLN SLQ NSFPL - GNTG DLW S TF H0 YA 5 03 FzdYMFT 1441 GRIIPI 529 CARGIHGD 1482 RASQAIG 1499 AAS 1175 CQQY 1541 0S 7GHD LGIAN YGLDYYYM RRLA SLQ DTYW - MH YA DVW S TF A0 6 02 Fzd YTFTG 379GWM 583 CARGMEY 1484 RASQGIS 1076 AAS 1179 CLQYN 1528 9S 7 YYMH NPNS WSYLA TLQ TYPW - GNTG S TF C1 YA 2 03 Fzd YTFTG 1450 GWM 1464 CARGPADF1485 RASQGIS 1504 DAS 1190 CQQS 1539 0S 7 YYIH DPNS WSGYKND SWLA SLQYSTPY - GYTG YFDFW S SF C0 YA 6 4A12 Fzd GYTFT 1439 WIYP 1469 CVRSAWG1496 KASQDV 1497 WA 1527 QQYS 1546 5 NYDI RDGS FAY GTAVA STR TYPLT NTKYN HT EKFK G

TABLE 1B Anti-Fzd Antibody Clone IDs, Heavy Chain (HC) and Light Chain(LC) Seq ID Nos, and Binding Characteristics Clone ID HC SID NO LC SIDNO Confirmed Binding 001S-B01 1 38 Fzd1, 2, 7, 9 001S-E02 2 39 Fzd1, 2,7 001S-G02 3 40 Fzd1, 2, 7 001S-H02 4 41 Fzd1, 2, 7 001S-A03 5 42 Fzd1,2, 7, 9 001S-B03 6 43 Fzd1, 2, 7 004S-G06 7 44 Fzd5, 8 002S-B01 8 Fzd1002S-C02 9 Fzd1 002S-E02 10 Fzd1 002S-G02 11 Fzd1 002S-F03 12 Fzd1002S-A04 13 Fzd1 002S-B04 14 Fzd1 002S-D04 15 Fzd1 004S-H04 16 45 Fzd5001S-A04 17 46 Fzd1, 2, 5, 7, 8 003S-E07 18 47 Fzd2 003S-D10 19 48 Fzd4004S-B08 20 49 Fzd6 004S-D08 21 50 Fzd6 004S-C09 22 51 Fzd6 004S-F10 2352 Fzd6 004S-A11 24 53 Fzd6 004S-Al2 25 54 Fzd6 005S-B07 26 55 Fzd9005S-D08 27 56 Fzd9 005S-E09 28 57 Fzd9 005S-H10 29 58 Fzd9 005S-B11 3059 Fzd9 005S-D11 31 60 Fzd9 014S-G02 32 61 Fzd6 014S-B04 33 62 Fzd8014S-B06 34 63 Fzd9 014S-G06 35 64 Fzd10 014S-A07 36 65 Fzd10 017S-B0937 Fzd8 004S-D01 129 130 Fzd4 004S-E09 131 132 Fzd6 004S-F09 Fzd6004S-H09 Fzd6 004S-B10 Fzd6 004S-C10 Fzd6 004S-F10 Fzd6 004S-G10 Fzd6004S-A11 Fzd6 004S-B11 n.b. 004S-D11 Fzd6 004S-E11 n.b. 004S-F11 Fzd6004S-G11 Fzd6 004S-A12 Fzd6 004S-B12 Fzd6 004S-C12 n.b. 004S-D12 n.b.004S-F12 n.b. 004S-F12 n.b. 004S-G12 n.b. 005S-B02 n.b. 005S-C02 n.b.005S-D02 Fzd5, 8 005S-E02 Fzd5, 8 005S-H02 Fzd5, 8 005S-A03 Fzd5, 8005S-C03 n.s. 005S-E03 n.s. 005S-F03 Fzd8 005S-B04 Fzd5, 8 005S-F04 n.b.005S-G04 Fzd5, 8 005S-H04 n.b. 005S-E05 n.b. 005S-G05 Fzd5, 8 005S-H05Fzd5, 8 005S-D06 Fzd8 005S-F06 n.b. 005S-G06 n.b. 005S-A07 Fzd9, 10005S-B07 Fzd9 005S-A08 Fzd9 005S-B08 Fzd9 005S-D08 Fzd9 005S-E08 Fzd9005S-F08 n.b. 005S-C09 Fzd9 005S-D09 Fzd9 005S-E09 Fzd9 005S-F09 Fzd9005S-A10 Fzd9 005S-B10 Fzd9 005S-E10 Fzd9 005S-H10 Fzd9 005S-B11 Fzd9005S-D11 Fzd9 005S-G11 n.b. 005S-H11 n.b. 005S-E12 Fzd10 006S-A01 Fzd10006S-H01 n.b. 006S-A02 Fzd10 006S-D02 n.b. 006S-H02 Fzd10 006S-A03 n.b.006S-B03 n.b. 006S-C03 n.b. 014S-A01 Fzd1, 2, 7 014S-B02 n.b. 014S-G02Fzd6 014S-B03 n.b. 014S-C03 Fzd1, 2, 7 014S-A04 n.b. 014S-B04 Fzd8014S-B05 Fzd5, 8 014S-B06 Fzd9 014S-F06 n.s. 014S-G06 Fzd10 014S-A07Fzd10 017S-E08 Fzd8 017S-H08 n.b. 017S-A09 n.b. 017S-B09 Fzd8 018S-F06Fzd4 018S-H06 n.b. 018S-B07 n.b. 017S-A10 n.b. 017S-B10 n.b. 017S-D10n.b. 018S-H08 n.b. 018S-B09 Fzd5, 8 021S-A01 n.b. 021S-E02 Fzd5, 8021S-G02 n.s. 021S-A03 n.b. 029S-B01 n.b. 029S-D01 Fzd1, 2, 7 029S-C02Fzd1, 2, 7 029S-H02 Fzd1 030S-A02 Fzd7 029S-E06 Fzd2, 6, 3 030S-F04 Fzd3030S-H05 Fzd7 030S-A06 Fzd1, 2, 7, 5 029S-C12 Fzd7 030S-C06 Fzd1001S-A01 Fzd1, 2, 7 001S-H01 Fzd1, 2, 7

In certain embodiment, the Fzd binding domain may be selected from anybinding domain that binds a Fzd with an affinity of, e.g., a K_(D) of atleast about 1×10⁻⁴ M, at least about 1×10⁻⁵ M, at least about 1×10⁻⁶ M,at least about 1×10⁻⁷ M, at least about 1×10⁻⁸ M, at least about 1×10⁻⁹M, or at least about 1×10⁻¹⁰ M. In certain embodiment, the Fzd bindingdomain may be selected from any binding domain that binds one or moreFzd receptor at high affinity, e.g., a K_(D) of less than about 1×10⁻⁷M, less than about 1×10⁻⁸ M, less than about 1×10⁻⁹ M, or less thanabout 1×10⁻¹⁰ M. In certain embodiment, the Fzd binding domain may beselected from any binding domain that binds Fzd at high affinity, e.g. aK_(D) of less than or equal to about 1×10⁻⁴ M, less than or equal toabout 1×10⁻⁵ M, less than or equal to about 1×10⁻⁶ M, less than or equalto about 1×10⁻⁷ M, less than or equal to about 1×10⁻⁸ M, less than orequal to about 1×10⁻⁹ M, or at least about 1×10⁻¹⁰ M in the context of aWnt surrogate molecule.

Suitable Fzd binding domains include, without limitation, de novodesigned Fzd binding proteins, antibody derived binding proteins, e.g.scFv, Fab, etc. and other portions of antibodies that specifically bindto one or more Fzd proteins; VHH or single domain antibody derivedbinding domains; knottin-based engineered scaffolds; norrin andengineered binding fragments derived therefrom, naturally occurring Fzdbinding domains, and the like. A Fzd binding domain may be affinityselected to enhance binding to a desired Fzd protein or plurality of Fzdproteins, e.g. to provide tissue selectivity.

In some embodiments, the Fzd binding domain binds to one, two, three,four, five or more different frizzled proteins, e.g. one or more ofhuman frizzled proteins Fzd1, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8,Fzd9, Fzd10. In some embodiments, the Fzd binding domain binds to Fzd1,Fzd2, and Fzd 7. In some embodiments, the Fzd binding domain binds toFzd1, Fzd2, Fzd5, Fzd7 and Fzd8. In other embodiments the Fzd bindingdomain is selective for one or more frizzled protein of interest, e.g.having a specificity for the one or more desired frizzled protein of atleast 10-fold, 25-fold, 50-fold, 100-fold, 200-fold or more relative toother frizzled proteins.

In certain embodiments, the Fzd binding domain comprises the six CDRregions of the pan specific frizzled antibody OMP-18R5 (vantictumab). Incertain embodiments, the Fzd binding domain is an scFv comprising thesix CDR regions of the pan-specific frizzled antibody OMP-18R5(vantictumab). See, for example, U.S. Pat. No. 8,507,442, hereinspecifically incorporated by reference. For example, the CDR sequencesof OMP-18R5 include a heavy chain CDR1 comprising GFTFSHYTLS (SEQ IDNO:270), a heavy chain CDR2 comprising VISGDGSYTYYADSVKG (SEQ IDNO:677), and a heavy chain CDR3 comprising NFIKYVFAN (SEQ ID NO:1033),and (ii) a light chain CDR1 comprising SGDKLGKKYAS (SEQ ID NO:1152) orSGDNIGSFYVH (SEQ ID NO:1153), a light chain CDR2 comprising EKDNRPSG(SEQ ID NO:1200) or DKSNRPSG (SEQ ID NO:1201), and a light chain CDR3comprising SSFAGNSLE (SEQ ID NO:1435) or QSYANTLSL (SEQ ID NO:1436). Inparticular embodiments, the Fzd binding domain is an antibody orderivative thereof, including without limitation scFv, minibodies, VHHor single domain antibodies (sdAb) and various antibody mimeticscomprising any of these CDR sequences. In certain embodiments, these CDRsequences comprise one or more amino acid modifications.

In other embodiments, the Fzd binding domain comprises a variable regionsequence, or the CDRs thereof, from any of a number of frizzled specificantibodies, which are known in the art and are commercially available,or can be generated de novo. Any of the frizzled polypeptides can beused as an immunogen or in screening assays to develop an antibody.Non-limiting examples of frizzled binding domains include antibodiesavailable from Biolegend, e.g. Clone CH3A4A7 specific for human frizzled4 (CD344); Clone W3C4E11 specific for human Fzd9 (CD349); antibodiesavailable from Abcam, e.g. ab64636 specific for Fzd7; ab83042 specificfor human Fzd4; ab77379 specific for human Fzd7; ab75235 specific forhuman Fzd8; ab102956 specific for human Fzd9; and the like. Otherexamples of suitable antibodies are described in, inter alia, US Patentapplication 20140105917; US Patent application 20130230521; US Patentapplication 20080267955; US Patent application 20080038272; US Patentapplication 20030044409; etc., each herein specifically incorporated byreference.

The Fzd binding region of a Wnt surrogate molecule may be an engineeredprotein that is selected for structural homology to the frizzled bindingregion of a wnt protein. Such proteins can be identified by screening astructure database for homologies. The initial protein thus identified,for example the microbial Bh1478 protein. The native protein is thenengineered to provide amino acid substitutions that increase affinity,and may further be selected by affinity maturation for increasedaffinity and selectivity in binding to the desired frizzled protein.Non-limiting examples of frizzled binding moieties include the Fz27 andFz27-B12 proteins.

In particular embodiments, a Wnt surrogate molecule comprises an LRP5/6binding domain, e.g., an anti-LRP5/6 antibody, or antigen-bindingfragment thereof, fused to a polypeptide that specifically binds to oneor more Fzd receptors. In particular embodiments, the polypeptide thatspecifically binds to LRP5/6 is an antibody or antigen-binding fragmentthereof. If certain embodiments, it is an antibody or antigen-bindingfragment thereof disclosed in the U.S. provisional patent applicationNo. 62/607,879, titled, “Anti-LR5/6 Antibodies and Methods of Use,”Attorney docket number SRZN-005/000S, filed on Dec. 19, 2017, which isincorporated herein by reference in its entirety. In particularembodiments, the LRP5/6 binding domain comprises the three heavy chainCDRs and/or the three light chain CDRs disclosed for any of theillustrative antibodies or fragments thereof that bind to LRP5 and/orLRP6 provided in Table 2A. In particular embodiments, the LRP5/6 bindingdomain comprises the three heavy chain CDRs and/or the three light chainCDRs disclosed for any of the illustrative antibodies or fragmentsthereof that bind to one or more Fzd receptor provided in Table 2A,wherein the CDRs collectively comprise one, two, three, four, five, six,seven, or eight amino acid modifications, e.g., substitutions,deletions, or additions. In certain embodiments, the LRP5/6 bindingdomain is a VHH or sdAb or was derived from a VHH or sdAb, so Table 2Aonly includes the three heavy chain CDRs. In certain embodiments, theLRP5/6 biding domain comprises the three heavy chain CDRs shown in Table2A or variants wherein the CDRs collectively comprise one, two, three,four, five, six, seven or eight amino acid modifications. In particularembodiments, the LRP5/6 binding domain comprises the heavy chainfragment and/or light chain fragment of any of the illustrativeantibodies or fragments thereof that bind to LRP5 and/or LRP6 providedin Table 2B or SEQ ID NOs:66-88 or 133 (or an antigen-binding fragmentor variant of either). In certain embodiments, the LRP5/6 binding domainis an Fab or was derived from an Fab, so Table 2B includes VH and CH1sequence, but not CH2 or CH3 sequences. In certain embodiments, theLRP5/6 binding domain is a VHH or sdAb or was derived from a VHH orsdAb, so Table 2B includes the VHH domain. In certain embodiments, theLRP5/6 binding region is a polypeptide, e.g., an antibody orantigen-binding fragment thereof, that competes with one of theseantibodies for binding to LRP5 and/or LRP6.

TABLE 2A Anti-LRP5/6 Antibody Clone IDs and CDR sequences. ConfirmedCDRH1 CDRH2 CDRH3 CDRL1 CDRL2 CDRL3 Clone ID Binding CDRH1 SEQ ID CDRH2SEQ ID CDRH3 SEQ ID CDRL1 SEQ ID CDRL2 SEQ ID CDRL3 SEQ ID 001S-CO8LRP6e1e2 YTISNYYIH 1682 GMINPSGGST 1762 CAIVRGKKWYFDLW 1842 RASQYISNYLN2098 AASSLQS 2110 CQQSYITPLTF 2160 TYA 001S-C10 LRP6e1e2 RTFGTYPNG 1632AAISWGGRTA 1700 CYARTVIGGFGAFRA 2061 YA HW 001S-D10 LRP6e1e2 RTFSRYAMA1642 AAIRWSGGGT 1689 CAASMEAMNSLRVN 1836 YYA KERYYQSW 001S-E10 LRP6e1e2LTFSNAAMA 1614 AAISRSGANTA 1696 CTLVNEIKTWW 2039 YS 001S-F10 LRP6e1e2RTFSSYAMA 1645 AAIKWSGTNT 1684 CAASMEAMNSLRVN 1834 YYA KERYYQSW 001S-G10LRP6e1e2 RTFSRYVMG 1644 AAITWRGGST 1706 CATGPNSIY 1987 YYA 001S-A11LRP6e1e2 RTFGNYDMG 1630 AGIRWSGSTL 1709 CYARTVIGGFGAFRA 2062 YA HW001S-B11 LRP6e1e2 RRFTTYGMG 1623 AAVTWRSGST 1708 CAAGSTVVAEFNYW 1828 YYA001S-C11 LRP6e1e2 SISSFNTMG 1659 AVITTGGDTSYS 1741 CNKVNAITKL 2025001S-E11 LRP6e1e2 RTLSRYSMG 1651 AAISRSGDRIY 1697 CTLVNEIKTWW 2040 YS001S-F11 LRP6e1e2 RTFSSYAMS 1646 AVIGRSGGIKY 1736 CATRRPFNSYNTEQS 1989YA YDSW 001S-G11 LRP6e1e2 SIFRLGTMY 1655 ASIGKSGSTNYA 1719 CKQHPNGYR2005 001S-H11 LRP6e1e2 RTLSSFAMG 1652 ATISRSGGNTY 1732 CNLREWNNSGAGY2026 YA W 001S-A12 LRP6e1e2 IAFRYYDMG 1608 AAITWNGRSS 1704CAAVFTGRFYGRPPR 1838 DYA EKYDYW 001S-B12 LRP6e1e2 RLLSYYALA 1622AAISRNGDKS 1694 CTLVNEIKTWW 2038 HYS 001S-C12 LRP6e1e2 RTFSNYAVG 1641AAISRFGGSTY 1693 CAADRIENYLGRYYD 1824 YV PSEYEYW 001S-D12 LRP6e1e2RFFSRYAMG 1643 GAISRSGNNT 1744 CTLVNEIKTWW 2041 YYA 001S-F12 LRP6e1e2RTFRSYTMG 1637 AAISGSGGSTT 1690 CNADIKTTTYSPLRNYW 2009 YA 008S-B01 LRP5TIFSINTMG 1664 ATMTSGGNT 1734 CYRRQWASSWGAR 2064 NYA NYEYW 008S-C01 LRP5NINSIETLG 1617 ANMRGGGY 1716 CHGRDYGSNAPQYW 2001 MKYA 008S-D01 LRP5NINSIETLG 1617 ANMRGGGY 1716 CYVKLRDDDYVYR 2065 MKYA 008S-E01 LRP5NINSIETLG 1617 ANMRGGGY 1716 CNAVTYNGYTIR 2023 MKYA 008S-G01 LRP5NINSIETLG 1617 ANMRGGGY 1716 CYARTQRMGVVNSYW 2060 MKYA 008S-A02 LRP5NINSIETLG 1617 ANMRGGGY 1716 CNAVTFGGNTIR 2021 MKYA 008S-C02 LRP5NINSIETLG 1617 ANMRGGGY 1716 CNAVTYDGY 2022 MKYA 008S-D02 LRP5 NINSIETLG1617 ANMRGGGY 1716 CAAQFRNDYGLRYQ 1832 MKYA STNNYW 008S-E02 LRP5NINSIETLG 1617 ANMRGGGY 1716 CNANYRGNRYW 2019 MKYA 009S-C01 LRP6e3e4GSFSGYYWT 1595 GEINHSGATN 1745 CVRYAWPEFDHW 2053 RASQRVSNY 2089 AASSLQG2110 CQQSYSVPYTF 2178 YN LN 009S-B02 LRP6e3e4 GSLSGYYWS 1596GEINHSGSTNYN 1746 CVRYAWPEFDHW 2055 RASQSISNYLN 2090 AASSLQS 2110CQQSYSLPLTF 2170 009S-C02 LRP6e3e4 GSFSDYYWS 1594 GEINHSGSTNYN 1746CVRYAWPEFDHW 2054 RASQSISNYLN 2090 AASSLQS 2110 CQQSYSMPLTF 2171009S-D02 LRP6e3e4 GTFSSYAIS 1603 GGIIPIFGTANYA 1749 CVYGRDFDYW 2056SGSSSNVGN 2105 DNDKRPS 2122 CESWDSSLSSE 2139 NYVS VF 010S-A02 LRP6e1e2HTFSSYAMG 1607 AAISQSGYVRYYA 1691 CKIYGLNGQPLGSW 2003 010S-B02 LRP6e1e2RTFNSGTMG 1634 AAITWRGGIT 1705 CNADGYSWDGRSG 2008 YYA RRLELW 010S-D02LRP6e1e2 RTFSSYAVG 1647 AAISYSGGSTK 1702 CAASVYISRRDSDYG 1837 YA YW010S-E02 LRP6e1e2 LSSGRPFSSY 1612 AAISWSGGST 1701 CKLQVRPIGYSSAYSR 2004VMG KYA NYW 010S-F02 LRP6e1e2 RSFNSYVIG 1625 AAIRWSGDNT 1688CAASMEAMNSLRVN 1835 YYA KERYYQSW 009S-E02 LRP6e1e2 RRFTTYGMG 1623AAVTWRSGST 1708 CAAGSTVVAEFNYW 1829 YYA 009S-F02 LRP6e1e2 RTFSYYAMG 1649AAISRSGGIYYA 1698 CNTVRPLWAW 2029 009S-G02 LRP6e1e2 SIFSIYAMG 1658AVITSGGKTVYA 1740 CYADSRSSWYDEYLE 2058 HW 009S-H02 LRP6e1e2 SIVRSLPMA1660 ATINDAQRYYA 1727 CNTSPYMHDVW 2027 009S-A03 LRP6e1e2 RTFSVYGVG 1648AAVSASGGYT 1707 CKAAPRWGGATAYW 2002 WYA 010S-G02 LRP6e1e2 SIVRSLPMA 1660ATINDAQRYYA 1727 CNTSPYMHDVW 2028 010S-A03 LRP6e1e2 RTFRRYAMG 1636ATISASGGNTA 1731 CNAPAWLYDDDYW 2020 YA 009S-B03 LRP6e1e2 RTFSNYAVG 1641AAISRFGGSTY 1693 CAADRIENYLGRYYD 1825 YV PSEYEYW 010S-B03 LRP6e1e2RTFSNYAVG 1641 AAISRFGGSTY 1692 CHAKQLRNGQMYTYW 1999 YA 010S-D03LRP6e1e2 ISSVYGMG 1609 AAIQWSADNT 1686 CAARTSGGLFHYRRS 1833 FYA DHWDTW009S-C03 LRP6e1e2 LPFSRYAMA 1610 AGMSGEGRN 1713 CSSRGYW 2034 TKYR009S-D03 LRP6e1e2 SIFSDGAMG 1656 AVISGGRTGYA 1737 CNTYPFPIYKKGYPFW 2030009S-E03 LRP6e1e2 RRFTTYGMG 1623 AAVTWRSGST 1708 CAAGSTVVAEFNYW 1830 YYA009S-F03 LRP6e1e2 RTFSSYAMS 1646 AVIGRSGGIKY 1736 CATRRPFNSYNTEQS 1990YA YDSW 010S-E03 LRP6e1e2 RSVSIYPMG 1628 AAINWSGDST 1685CNAVVVGLSRRIDNIW 2024 KYA 010S-F03 LRP6e1e2 RTFSRYVMG 1644 AAITWRGGST1706 CATGPNSIY 1988 YYA 009S-G03 LRP6e1e2 RSVSSYNMG 1629 AAISRRGGIIEYG1695 CHAVENILGRFVDYW 2000 009S-H03 LRP6e1e2 SIFSINTMG 1657 AVITSGGKTVYA1740 CYADSRSSWYDEYLE 2057 HW 009S-A04 LRP6e1e2 RTLSAYDMG 1650 GGIRWSGGTT1753 CYARTVIGGFGAFRA 2063 LYP HW 009S-B04 LRP6e3e4 SIFMINTMA 1654ATIRPVVSETT 1728 CNAKRPWGTRDEYW 2018 YA 010S-G03 LRP6e3e4 RSFNSYTTT 1624AAIRGSSGSTF 1687 CNAASTVTAWPYYG 2006 YA PDYW 009S-C04 LRP6e3e4 FRFSISTMG1553 AYITGGGRTM 1743 CNAFVRSDFDRYYDYW 2011 DG 009S-D04 LRP6e3e4TIVSIYRIN 1665 AGITSSGRTIYA 1712 CNAASTVTAWPYYG 2007 PDYW 010S-H03LRP6e3e4 RIFSIYDMG 1621 SGIRWSGGTS 1789 CSSRGYW 2035 YA 009S-E04LRP6e3e4 RIFAIYDIA 1618 AMIRPVVTEID 1715 CNAKRPWGSRDEYW 2012 YA 010S-A04LRP6e3e4 SLFSFNAVG 1662 ASISSGGRTNYA 1722 CSKGGVYGGTYVPD 2032 SW009S-F04 LRP6e3e4 RSLSSFAMG 1627 ARISRGDGYT 1718 CAAVQAVIGGTLTTA 1839DEA YDYW 010S-B04 LRP6e3e4 RVLSYYAMA 1653 AGITRGGATTY 1711CAAGPNWSTRNREY 1827 YS DYW 009S-G04 LRP6e3e4 GTFSRYHMG 1601 SAITWSGGRT1788 CALTWAPTPTNRRS 1872 YYA DYAYW 009S-H04 LRP6e3e4 RIFAIYDMA 1619ATIRPVVSETT 1728 CNAKRPWGTRDEYW 2017 YA 010S-C04 LRP6e3e4 SLFSFNAMG 1661ASISSGSRTNYA 1723 CSKGGVYGGTYVPD 2033 SW 010S-D04 LRP6e3e4 RIFAIYDIA1618 ATIRPVVTQID 1730 CNAKRPWGSRDEYW 2015 YA 010S-E04 LRP6e3e4 RTFGSDVMG1631 ALTGWGDGS 1714 CAAARRSGTYDIGQY 1820 TTYYE LRESAYVFW 010S-F04LRP6e3e4 RTFSRYAMG 1643 AAITRSGSNTY 1703 CAADPRGVTLPRATA 1823 YA YEYW009S-A05 LRP6e3e4 RTFSDYSMG 1639 AGISWIADNR 1710 CTAGRSRYLYGSSLN 2036YYA GPYDYW 010S-G04 LRP6e3e4 VIFALYDIA 1666 ATIRPVVTETD 1729CNAKRPWGSRDEYW 2014 YA 010S-H04 LRP6e3e4 RSFSDFFMG 1626 ATISWSGSSA 1733CAAAYSYSQYGSSYS 1821 NYE YW 010S-A05 LRP6e3e4 LSFSSYAMG 1611 AAISRSGVSTY1699 CAAKFGVLATTESRH 1831 YA DYW 010S-C05 LRP6e3e4 RTFNIDDMG 1633ASIRWSGQSP 1720 CNAETYSGNTIW 2010 YYA 010S-D05 LRP6e3e4 RTFSDYSMA 1638AGISWIADNR 1710 CAGDRSRYLYGDSLR 1841 YYA GPYGYW 010S-E05 LRP6e3e4SVFTTFAKG 1663 ASITASSDRTF 1725 CAAYSTFNTDVASM 1840 YA KPDYW 010S-F05LRP6e3e4 RIFSIYDIA 1620 ATIRPVVTETD 1729 CNAKRPWGSRDEYW 2013 YA 013S-G04LRP6e3e4 RIFAIYDIA 1618 ATIRPVVSETT 1728 CNAKRPWGTRDEYW 2016 YA 013S-H04LRP6e3e4 RFSMYDMG 1640 ASIRWSSGNT 1721 CYANIYYTRRAPEEYW 2059 WYA013S-A05 LRP6e3e4 RTFNTYAMG 1635 ASVSWRYDRT 1726 CAADTNWRAGPRV 1826 YYTGIDEYAYW 013S-B05 LRP6e3e4 FAFSTTAMS 1549 STINPGGLSKS 1806 CTKGGIQ 2037YA 013S-C05 LRP6e3e4 NIFPIDDMS 1616 ATVTSGGRINYA 1735 CNVDRTLYGKYKEYW2031 013S-D05 LRP6e3e4 RIFSIYDMG 1621 SGIRWSGGTS 1789 CGSRGYW 1998 YA013S-E05 LRP6e3e4 YTFTYRYLH 1681 GGIIPIFGTADYA 1748 CARDWELYGMDVW 1907RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 013S-F05 LRP6e3e4GTFSSYAIS 1603 GIINPSGGSTSYA 1761 CARAGYYDSSGYYAF 1882 RASQSISSYLN 2094AASSLQS 2110 CQQSYSTPLTF 2174 YA DIW 013S-G05 LRP6e3e4 YTFTYRYLH 1681GGVIPIFGTAD 1755 CASDIVVDDAFDTW 1969 RASQDISNY 2079 AASTLQS 2113CQQGNSFPYTF 2152 YA LN 010S-G06 LRP6e3e4 FSFETYGMS 1555 SGISGSGGRTH 1792CARDLDYW 1897 QASQDISNY 2077 AASSLQS 2110 CQQSYRIHWTF 2163 YA LN009S-B05 LRP6e3e4 FTFDAYAMH 1560 STLSGDANNA 1811 CARGGSGWSNYYG 1931RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YYA MDVW 009S-C05LRP6e3e4 YTFTYRYLH 1681 GRIIPVLKITNYA 1768 CAVVDDAFDIW 1996 009S-D05LRP6e3e4 FTLRNHWLS 1591 SAISGSGGSTY 1786 CATRTGYSYGFNFW 1991 RASQSISSYLN2904 AASSLQS 2110 CQQSYSTPLTF 2174 YA AFDIW 009S-E05 LRP6e3e4 YTFTNNFMH1676 GHVDPGDGET 1756 CARDWGIAAAGDYY 1908 RASQGINSY 2081 DAKGLHP 2114CQQSYSAPLSF 2166 IYA YYGMDVW LA 009S-F05 LRP6e3e4 FTFDDYGMS 1561SAIGTGGGTYYA 1781 CARLGSYGSPYYYYG 1959 RASQSISSYLN 2094 AASSLQS 2110CQQSYSTPLTF 2174 MDVW 009S-G05 LRP6e3e4 FTFSDYYMS 1568 SGVSWNGSRT 1799CAKDSGLV 1852 QASQDISNY 2077 AASTLQR 2112 CQQSYSAPLTF 2167 HYA LN009S-C06 LRP6e3e4 YTFASYDIH 1671 GWMNPNSG 1776 CARATGSGWYTDLG 1883RASRNINRY 2099 AASSLLS 2109 CQQSYNVPFTF 2162 NTGYA YW LN 009S-D06LRP6e3e4 FTFSSHSTH 1573 STISDTNSGTY 1807 CAKAQATGWSGYYT 1843 RASQSISSYLN2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA FDYW 009S-E06 LRP6e3e4 FTFTDYGLH1587 AVISYGGSNKY 1739 CASGYSYGLYYYGM 1973 RASQSISSYLN 2094 AASSLQS 2110CQQSYSTPLTF 2174 YA DVW 009S-F06 LRP6e3e4 YTFTYRYLH 1681 GGIIPIFGTAN1749 CATEAALDAFDIW 1985 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174YA 009S-G06 LRP6e3e4 YIFTDYYMH 1669 GWINPNSGGT 1774 CARDFLGSTGDYW 1892RASQNIGLY 2088 DASSLQR 2121 CQQSYSTPYTF 2176 NYA LN 009S-H06 LRP6e3e4FTFSSSAMH 1574 SAIGTGGSTYYA 1783 CAKGGDYFYYYYGM 1856 RASQSISSYLN 2094AASLQS 2110 CQQSYSTPLTF 2174 DVW 009S-A07 LRP6e3e4 YTFTYRYLH 1681GGIIPIFGTAN 1749 CATAYGSSSLNIDYW 1980 RASQSISSYLN 2094 AASSLQS 2110CQQSYSTPLTF 2174 YA 009S-B07 LRP6e3e4 YTFTGYYMH 1675 GWINPNSGGT 1774CVKDGGSFPLAYAFD 2049 RASQSISSYLN 2904 AASLQS 2110 CQQSYSTPLTF 2174 NYAIW 009S-D07 LRP6e3e4 FPFRYYGMS 1551 ARIGWNGGSI 1717 CARDYSDRSGIDYW 1910RSSQSLLHS 2102 LGSNRAS 2131 CMQATQFPLTF 2146 VYA NGYNYLD 009S-F07LRP6e3e4 GTFSSYAIS 1603 GIINPSGGSTS 1761 CARAAGNFWSGYYT 1876 RASQSISSYLN2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA FDYW 009S-G07 LRP6e3e4 YTFTYRYLH1681 GGIIPIFGTAN 1749 CARGSYGMDVW 1947 RASQGISNY 2083 DASNLET 2115CLQDFSFPWTF 2140 YA LA 009S-H07 LRP6e3e4 YTFTGYYMH 1675 GWMNPNSG 1776CASSVVPAGPAGVY 1975 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 NTGYAAFDIW 009S-A08 LRP6e3e4 GTFSSHAIN 1602 GWISANNGN 1775 CARDQDYGDYGWY 1902RASQGISNY 2083 GSSTLQS 2127 CQQTYSIPPTF 2181 TDYA YYGMDVW LA 011S-C01LRP6e3e4 LTFTSHGMS 1615 SYVSDSGSSVY 1818 CARHPGSFGGYSYA 1956 RASQSISSYLN2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA WYYYYGMDVW 009S-C08 LRP6e3e4FSFNTFGIH 1556 AVISYDGSNKY 1738 CAKSIAAAGTGYYG 1868 RASQSISSYLN 2094AASSLQS 2110 CQQSYSTPLTF 2174 YA MDVW 009S-D08 LRP6e3e4 YTFTSYDIN 1679GGIIPIFGTAN 1749 CARGPYYFDYW 1939 RASQGISNN 2082 DASSLES 2120CLQHNSYPFTF 2143 YA LN 011S-F01 LRP6e3e4 FSFSDYYMS 1558 SGISESGGRTY 1790CASAADFDYW 1966 RASQDISNY 2079 AASSLQS 2110 CLQDYSYPRTF 2141 YA LN009S-E08 LRP6e3e4 TGFTGYYIH 1668 GWMNPNSG 1776 CARGYGDYDLW 1951QASQDISNY 2077 DASSLES 2120 CQQSYRYPTF 2165 NTGYA LN 009S-F08 LRP6e3e4DTFANYGFS 1547 GXVNAGNGN 1777 CAKGWLDFDYW 1866 QASQDISNY 2077 DASSLES2120 CQQSYSTSITF 2177 TTYA LN 009S-G08 LRP6e3e4 FTFSDFAMT 1566SYISGDSGYTN 1813 CARLGSYPGPYYYYM 1961 RASQSISSYLN 2094 AASSLQS 2110CQQSYSTPLTF 2174 YA DVW 009S-H08 LRP6e3e4 YTFTDYFMN 1673 GIINPSGDSTR1758 CARDDGLGGMDVW 1888 QASQDISNY 2076 AASSLQS 2110 CQQSYSTPLTF 2174 FALA 009S-A09 LRP6e3e4 YTFTYRYLH 1681 GRIIPILGSTNYA 1767 CTTDLWDYW 2047QASQGITNY 2078 AASSLQS 2110 CLQDYTDPFTF 2142 LN 011S-F02 LRP6e3e4FTFSTYGMH 1584 SSISVSSGTTH 1804 CARGGSGSYYYAFDIW 1929 RASQSISSYLN 2094AASSLQS 2110 CQQSYSTPLTF 2174 YA 011S-G02 LRP6e3e4 YTFTSYAMN 1678GGIIPIFGTAN 1749 CARDASGGSTGWYY 1886 RASQGISSYLA 2087 AASSLQS 2110CQQAYSFPWTF 2150 YA FDSW 011S-A03 LRP6e3e4 FTFSSYWMH 1580 STISGSGGRTY1808 CATSPYGVFTLDYW 1993 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174YA 011S-C03 LRP6e3e4 YTFSYRYLH 1672 GGIIPIFGTAN 1749 CASTVTTDAFDIW 1977QASQDISNY 2077 DASSLES 2120 CQQSYSFPPFTF 2168 YA LN 011S-D03 LRP6e3e4FSFDDYGMS 1554 SVISSGGTIYYA 1812 CARHLSSGYLSYYGM 1954 RASQSISSYLA 2093AASTLQS 2113 CQQSYSTPLTF 2174 DVW 011S-F03 LRP6e3e4 FTFSSYAMS 1577SAISGSGGSTY 1786 CAKGGRDGYKGYFD 1859 KSSQSVLYTT 2073 WASSRKS 2135CQQYYSTPYTF 2189 YA YW TNRNHIA 011S-C04 LRP6e1e2 GTFNSNAIS 1598 GWMNPNSG1776 CARDYYGSGSYNYG 1912 GASQSVPR 2066 GASQRAT 2124 CQQYHNWPP 2184 NTGYAMDVW NSLA EYTF 011S-D04 LRP6e1e2 YTFTSYDIN 1679 GIINPSGGSTS 1761CAREAYYYYYGMDVW 1915 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA011S-H04 LRP6e1e2 YIFTDYYMH 1669 GRIIPILGRANYA 1765 CARGGYSTLDYW 1932QASQDISNY 2077 AASTLQS 2113 CQQSFSTPRTF 2156 LN 008S-F02 LRP5 YTFTNYCMH1677 GIINPSDGSTS 1757 CAKDMVHLIVALAID 1851 RSSQSLLHS 2101 TLSYRAS 2134CMQALEALFTF 2144 HA YW DGYTYLY 010S-C06 LRP6e1e2 FTFNSYSMD 1563SSISPRGGSTY 1802 CAPYYYDKSAKPLRSY 1875 RASQSISSYLN 2094 AASSLQS 2110CQQSYSTPLTF 2174 YA FDHW 010S-E06 LRP6e3e4 LTVSSNYMS 1615 SGISWNSGSIG1796 CARGSDCSGGSCYYS 1944 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174YA FDYW 010S-F06 LRP6e3e4 FTFSSSWMH 1575 SAIGTGGGTYYA 1781CAREVAVKDYYYYY 1921 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 MDVW010S-H06 LRP6e3e4 YTFTSYDIN 1679 GRIIPILGRTNYA 1766 CAREERGATGRAFDIW1918 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 010S-A07 LRP6e3e4FTFSSYAMH 1576 ASISSTSGSKYYA 1724 CAKTYYDFWSGYYTF 1870 RASQSISSYLN 2094AASSLQS 2110 CQQSYSTPLTF 2174 DYW 010S-B07 LRP6e3e4 FTFSDYYMS 1568SMISYNGGRA 1800 CARGNPYYFDYW 1937 RASQSISSYLN 2094 AASSLQS 2110CQQSYSTPLTF 2174 FYA 010S-C07 LRP6e3e4 FTFSKTDMH 1569 STITTDSRGTY 1810CAKGGDYYYYYYGM 1858 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YADVW 010S-D07 LRP6e3e4 YTFTYRYLH 1681 GGIIPIFGTAN 1749 CANGLEDAYAFDIW1873 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA 009S-D05 LRP6e3e4FTLRNHWLS 1591 SAISGSGGSTY 1786 CATRTGYSYGFNFW 1992 RASQSISSYLN 2094AASSLQS 2110 CQQSYSTPLTF 2174 YA AFDIW 009S-E05 LRP6e3e4 YTFTNNFMH 1676GHVDPGDGET 1756 CARDWGIAAAGDYY 1909 RASQGINSY 2081 DAKGLHP 2114CQQSYSAPLSF 2166 IYA YYGMDVW LA 010S-E07 LRP6e3e4 YTFTYRYLH 1681GGIIPIFGTAN 1749 CAKDDFSLYGMDVW 1845 RASQSISSYLN 2094 AASSLQS 2110CQQSYSTPLTF 2174 YA 009S-F05 LRP6e3e4 FTFDDYGMS 1561 SAIGTGGGTYYA 1781CARLGSYGSPYYYYG 1960 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 MDVW010S-F07 LRP6e3e4 YTFTYRYLH 1681 GGIIPIFGTAN 1749 CARLDYGETEGNGDW 1958RASQSVYSN 2097 DTSNRAT 2123 CQQYNNWPP 2185 YA LA ITF 010S-G07 LRP6e3e4FTFSSYAMH 1576 STISGSGGSTY 1809 CARAGYGRYYYGMD 1880 RVSQGISSYLN 2103AASSLQS 2110 CQQTYTIPFTF 2182 YA VW 009S-G05 LRP6e3e4 FTFSDYYMS 1568SGVSWNGSRT 1799 CAKDSGLV 1853 QASQDISNY 2077 AASTLQR 2112 CQQSYSAPLTF2167 HYA LN 010S-H07 LRP6e3e4 YTFTYRYLH 1681 GGIIPIFGTAN 1749CARDDSMGAFDIW 1890 QASQDISNY 2077 GTSNLQS 2128 CQQSYSTPYTF 2176 YA LN010S-A08 LRP6e3e4 HTFLTYDIN 1606 GRITPRLGIAN 1770 CASYFGVMDVW 1979RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA 009S-A07 LRP6e3e4YTFTYRYLH 1681 GGIIPIFGTAN 1749 CATAYGSSSLNIDYW 1981 RASQSISSYLN 2094AASSLQS 2110 CQQSYSTPLTF 2174 YA 009S-B07 LRP6e3e4 YTFTGYYMH 1675GWINPNSGGT 1774 CVKDGGSFPLAYAFD 2050 RASQSISSYLN 2094 AASSLQS 2110CQQSYSTPLTF 2174 NYA IW 009S-B06 LRP6e3e4 YTFTYRYLH 1681 GGIIPIFGTAN1749 CAPALTDAGSFDYW 1874 RVSQSISSYLN 2103 AASSLQS 2110 CQQSYSTPLTF 2174YA 010S-B08 LRP6e3e4 YTFTYRYLH 1681 GGIIPVFGTAD 1751 CARDREQQILDYW 1904RASQGISNN 2082 DASNLET 2115 CQQSYTSRLTF 2179 YA LN 010S-C08 LRP6e3e4FTFSTFGMH 1582 STITSSGGSTYYA 1809 CARAGIAAAPGSRNY 1878 RASQSISSYLN 2094AASSLQS 2110 CQQSYSTPLTF 2174 YGMDVW 009S-C06 LRP6e3e4 YTFASYDIH 1671GWMNPNSG 1776 CARATGSGWYTDLG 1884 RASRNINRY 2099 AASSLLS 2109CQQSYNVPFTF 2162 NTGYA YW LN 009S-D06 LRP6e3e4 FTFSSHSTH 1573STISDTNSGTY 1807 CAKAQATGWSGYYT 1844 RASQSISSYLN 2094 AASSLQS 2110CQQSYSTPLTF 2174 YA FDYW 010S-D08 LRP6e3e4 FTFSSSWMH 1575 SAIGTGGGTYYA1781 CAKEDYDSSGYYYYY 1855 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174FQHW 009S-E06 LRP6e3e4 FTFTDYGLH 1587 AVISYGGSNKY 1739 CASGYSYGLYYYGM1974 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA DVW 010S-E08LRP6e3e4 YSFTRTDMH 1670 GYISAYTGHTS 1778 CARDLGGTADYW 1898 RASQSISSYLN2094 ZASSLQS 2137 CQQSYSTPLTF 2174 YA 010S-F08 LRP6e3e4 LTFDDHAMH 1613SYISSSGRTIFYA 1815 CVRGDSGWGILYYV 2052 RASQSISSYLN 2094 AASSLQS 2110CQQSYSTPLTF 2174 MDVW 009S-F06 LRP6e3e4 YTFTYRYLH 1681 GGIIPIFGTAN 1749CATEAALDAFDIW 1986 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA010S-G08 LRP6e3e4 YIFTDYYMH 1669 GGFDPEDGET 1747 CARGGGPNEHDYYF 1927RASQSVRSS 2096 GSSSRAT 2126 CQQYGRSPRY 2183 IYA DYW DLA SF 010S-H08LRP6e3e4 FTFZNAWMS 1590 SGISGSGGSTY 1793 CARGRGKKNYYYGM 1942 RASQSISSYLN2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA DVW 010S-A09 LRP6e3e4 FTFSTYYMS1586 SGISWNGGKT 1794 CARGGDFDYW 1925 QASQDIANY 2075 AASSLQS 2110CQQSYSTPYTF 2176 HYV LN 010S-B09 LRP6e3e4 GTFSSYAIS 1603 GWINPNSGDT 1773CARGEQWLVWGFD 1924 RASQSISRYLN 2092 KASSLES 2130 CQQSYDSPWTF 2159 NYA PW009S-G06 LRP6e3e4 YIFTDYYMH 1669 GWINPNSGGT 1774 CARDFLGSTGDYW 1893RASQNIGLY 2088 DASSLQR 2121 CQQSYSTPYTF 2176 NYA LN 010S-C09 LRP6e3e4YTFTYRYLH 1681 GGIIPIFGTAN 1749 CARDEVEGGMDVW 1891 RASQSISSYLN 2094AASSLQS 2110 CQQSYSTPLTF 2174 YA 009S-H06 LRP6e3e4 FTFSSSAMH 1574SAIGTGGSTYYA 1783 CAKGGDYFYYYYGM 1857 RASQSISSYLN 2094 AASSLQS 2110CQQSYSTPLTF 2174 DVW 010S-D09 LRP6e3e4 GTFSSYTIS 1603 GGIVPAYRRA 1754CAKGGYELDYW 1865 QASQDISNY 2077 AASSLQS 2110 CQQIHSYPLTF 2155 NYA LN010S-E09 LRP6e3e4 GDLSIYTIN 1593 GWINAGNGN 1772 CARGGDSSGYYYYAF 1926RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 TTYA DIW 009S-A07LRP6e3e4 YTFTYRYLH 1681 GGIIPIFGTAN 1749 CATAYGSSSLNIDYW 1982RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA 009S-B07 LRP6e3e4YTFTGYYMH 1675 GWINPNSGGT 1774 CVKDGGSFPLAYAFD 2051 RASQSISSYLN 2094AASSLQS 2110 CQQSYSTPLTF 2174 NYA IW 009S-D08 LRP6e3e4 YTFTSYDIN 1679GGIIPIFGTAN 1749 CARGPYYFDYW 1940 RASQGISNNLN 2082 DASSLES 2120CLQHNSYPFTF 2143 YA LN 010S-F09 LRP6e3e4 FTFDEYAMH 1562 STISGSGGSTY 1809CASAKNDFWSGYFA 1968 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YAFDYW 010S-G09 LRP6e3e4 GTFNTHTIT 1599 GWMNPNSG 1776 CARGNLDFDYW 1936QASQDISNY 2077 DASNLET 2115 CQQSYSTPLTF 2174 NTGYA LN 010S-H09 LRP6e3e4FTFSDHYMS 1567 SAISSGSDRTY 1787 CARYSGYDFDYW 1965 RASQGISNY 2084 AASTLQS2113 CQQGYGTPP 2153 YA LN MF 010S-A10 LRP6e3e4 FSFSSYSMN 1559SYISSSSSTIYYA 1816 CARGSGYYGPGYYG 1946 RASQSISSYLN 2094 AASSLQS 2110CQQSYSTPLTF 2174 MDVW 009S-D07 LRP6e3e4 FPFRYYGMS 1551 ARIGWNGGSI 1717CARDYSDRSGIDYW 1911 RSSQSLLHS 2102 LGSNRAS 2131 CMQATQFPLTF 2146 VYANGYNYLD 010S-B10 LRP6e3e4 FAFKDYYMT 1548 SAIGAGGGTY 1779 CARESALYSSSWYYY1920 RASQSISSYLN 2094 GTSSLHT 2129 CQQANSFPFTF 2148 YA YYGMDVW 010S-C10LRP6e3e4 FTFSSYAMS 1577 SAISGSGGSTY 1786 CAKGGRDGYKGYFD 1860 KSSQSILSSS2072 WASSRKS 2135 CQQYYNIPYSF 2187 YA YW SNRDSLA 009S-E07 LRP6e3e4YTFTGYYIH 1674 ZHVDPEDGETI 1819 CARGPAAIGILGWFD 1938 RASQSISSYLN 2094AASSLQS 2110 CQQSYSTPLTF 2174 YA PW 010S-D10 LRP6e3e4 YIFTDYYMH 1669GWMNPNSG 1776 CARTLSGYSSSWYVF 1964 RASQSISSYLN 2094 AASSLQS 2110CQQSYSTPLTF 2174 NTGYA DYW 010S-E10 LRP6e3e4 FTFSSYSMN 1579 SGISWNSGTT1797 CARDHSSGWRHYFD 1895 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174GYS YW 010S-F10 LRP6e3e4 FTFSNSDMN 1570 SYISGNSGYTN 1814 CASGSYYSDFDYW1971 RASQSISNYLN 2090 AASTLES 2111 CQQANSFPPTF 2148 YA 010S-G10 LRP6e3e4GTFSSYAIS 1603 GRINPNGGGT 1769 CAREGGYYFDYW 1919 RASQGISNY 2083 AASSLQS2110 CQQSYSTPWTF 2175 IYA LA 009S-F07 LRP6e3e4 GTFSSYAIS 1603GIINPSGGSTS 1761 CARAAGNFWSGYYT 1877 RASQSISSYLN 2094 AASSLQS 2110CQQSYSTPLTF 2174 YA FDYW 009S-G07 LRP6e3e4 YTFTYRYLH 1681 GGIIPIFGTAN1749 CARGSYGMDVW 1948 RASQGISNY 2083 DASNLET 2115 CLQDFSFPWTF 2140 YA LA010S-H10 LRP6e3e4 YTFTSYYMH 1680 GWINPNSGGT 1774 CAREAAEIPVGAFDIW 1914KSSHSLLYSS 2071 WSSTRES 2136 CQQYYSTPQTF 2188 NYA DNKNYLA 010S-A11LRP6e3e4 FTFSNSDMN 1570 SYISGNSGYTN 1814 CASGSYYSDFDYW 1972 RASQSIZNYLN2095 ZASTLES 2138 CQQANSFPPTF 2148 YA 010S-B11 LRP6e3e4 FTFRNYAIH 1564SAIGTGGDTYYA 1780 CARDGGIRDFDYW 1894 QASQDISNY 2077 AASTLQS 2113CQQSYSTPLTF 2174 LN 010S-C11 LRP6e3e4 YTFTYRYLH 1681 GGIIPIFGTAN 1749CAADDLGLELHYW 1822 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA009S-H07 LRP6e3e4 YTFTGYYMH 1675 GWMNPNSG 1776 CASSVVPAGPAGVY 1976RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 NTGYA AFDIW 009S-A08LRP6e3e4 GTFSSHAIN 1602 GWISANNGN 1775 CARDQDYGDYGWY 1903 RASQGISNY 2083GSSTLQS 2127 CQQTYSIPPTF 2181 TDYA YYGMDVW LA 010S-D11 LRP6e3e4YTFTYRYLH 1681 GGIIPVFGTAN 1752 CATDEYSSSYAFDIW 1983 RASQSVSSN 2097GASTRAT 2125 CQQFDRSPLTF 2151 YA LA 010S-E11 LRP6e3e4 FTFSAHGMH 1565SGISESGGSTY 1791 CARGRGYSYGYYAFD 1943 RASQSISSYLN 2094 AASSLQS 2110CQQSYSTPLTF 2174 YA IW 010S-F11 LRP6e3e4 YTFTYRYLH 1681 GGIIPIFGTAN 1749CARDSDWGVVDPW 1905 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA010S-G11 LRP6e3e4 YTFTYRYLH 1681 GRIIPVLKITNYA 1768 CAVVDDAFDIW 1997RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 010S-H11 LRP6e3e4YTFTYRYLH 1681 GGIIPIFGTAN 1749 CAKDGTDGRFDPW 1846 RASQDISSYLA 2080SASTLQS 2133 CQQSNSFPYTF 2157 YA 009S-B08 LRP6e3e4 FTFTSSAVQ 1589GWINAGNGN 1772 CARRGGDVTVPAAY 1963 RASQSISSYLN 2094 ZASSLQS 2137CQQSYSTPLTF 2174 TTYA YAMDVW 010S-A12 LRP6e3e4 VTFSRYPIS 1667GGIIPIFGTAN 1749 CAKDSGNYGYYGMD 1854 RASQSISSYLN 2094 AASSLQS 2110CQQSYSTPLTF 2174 YA VW 010S-B12 LRP6e3e4 FTFSSYDMH 1578 SGITSNGGATY 1798CARGTTGKGYYYYG 1949 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YAMDVW 010S-C12 LRP6e3e4 FTFSNYWIH 1571 SAIGTGGGTYYA 1781 CTTAGYKAARRSVYP2044 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 RIFNFDYW 010S-D12LRP6e3e4 YTFTYRYLH 1681 GRIIPIFGTANYA 1763 CAREEGVGGMDVW 1917 PRSQSIGSW2100 DASNLQS 2116 CQQSSSTPYTF 2158 LA 010S-E12 LRP6e3e4 FTFSSYAMH 1576SAIGAGGGTY 1779 CARGVSSGYYYYYG 1950 RASQSISSYLN 2094 AASSLQS 2110CQQSYSTPLTF 2174 YA MDVW 010S-F12 LRP6e3e4 FTVSSNYMS 1592 SAIGTGGGTYYA1781 CARAGTNWGGWYF 1879 RASQGISRD 2085 AASTLQS 2113 CQQSYSPPFTF 2172 DLWLA 010S-G12 LRP6e3e4 FALSGYYMS 1550 SSISSSSTYIRYA 1803 CATVTGYSSAGAFDIW1995 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 011S-A01 LRP6e3e4FTFSTHAFH 1583 SAIRGSGERTY 1784 CARDLRNWGSPYW 1901 RASQSISSYLN 2094AASSLQS 2110 CQQSYSTPLTF 2174 YA YFDLW 011S-B01 LRP6e3e4 GTFSHYTIS 1600GWINAGNGN 1771 CAKGGSLDMDVW 1864 RASQGISNY 2083 AASSLHS 2108 CQQSYRTPLTF2164 TKYS LA 011S-C01 LRP6e3e4 LTFTSHGMS 1615 SYVSDSGSSVY 1818CARHPGSFGGYSYA 1957 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YAWYYYYGMDVW 011S-D01 LRP6e3e4 GTISDYTVS 1605 GIINPSGGSTS 1761 CARGYYDFDYW1953 RASQGISNY 2083 AASSLQS 2110 CQQSYSTPLTF 2174 YA LA 009S-C08LRP6e3e4 FSFNTFGIH 1556 AVISYDGSNKY 1738 CAKSIAAAGTGYYG 1869 RASQSISSYLN2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA MDVW 011S-E01 LRP6e3e4 FPFZYYSMN1552 SAISGRDGRTY 1785 CAKDLGIQLPDYYFD 1847 RASQGISSA 2086 AASTLQS 2113CQQSYSSPPTF 2173 YA YW LA 009S-D08 LRP6e3e4 YTFTSYDIN 1679 GGIIPIFGTAN1749 CARGPYYFDYW 1941 RASQGISNN 2082 DASSLES 2120 CLQHNSYPFTF 2143 YA LN011S-F01 LRP6e3e4 FSFSDYYMS 1558 SGISESGGRTY 1790 CASAADFDYW 1967RASQDISNY 2079 AASSLQS 2110 CLQDYSYPRTF 2141 YA LN 009S-E08 LRP6e3e4YGFTGYYIH 1668 GWMNPNSG 1776 CARGYGDYDLW 1952 QASQDISNY 2077 DASSLES2120 CQQSYRYPTF 2165 NTGYA LN 009S-F08 LRP6e3e4 DTFANYGFS 1547 GXVNAGNGN1777 CAKGWLDFDYW 1867 QASQDISNY 2077 DASSLES 2120 CQQSYSTSITF 2177 TTYALN 011S-G01 LRP6e3e4 YTFTYRYLH 1681 GGIIPLFGTAN 1750 CTTDDYGDQYGMD 2046RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA VW 011S-H01 LRP6e3e4YTFTYRYLH 1681 GGIIPIFGTAN 1749 CTTDDYGDLTHLDYW 2045 RASQSISSYLN 2094AASSLQS 2110 CQQSYSTPLTF 2174 YA 011S-A02 LRP6e3e4 GTFSSYAIS 1603GWMNPNSG 1776 CARDKGYAFDIW 1896 RSSQSLLHS 2102 AASSLQS 2110 CMQALQTPITF2145 NTGYA NGYNYLD 011S-B02 LRP6e3e4 YSFTRTDMH 1670 GYISAYTGHTS 1778CARDLGGTADYW 1899 RZSQSZSZYLN 2104 AASSLQS 2110 CQQSYSTPLTF 2174 YA011S-C02 LRP6e3e4 FTFSTYSMN 1585 SGISWNSGRI 1795 CARDVGAFDIW 1906QASQDISNY 2077 AASILQS 2107 CQQSYSIPFTF 2169 GYA LN 009S-G08 LRP6e3e4FTFSDFAMT 1566 SYISGDSGYTN 1813 CARLGSYPGPYYYYM 1962 RASQSISSYLN 2094AASSLQS 2110 CQQSYSTPLTF 2174 YA DVW 011S-D02 LRP6e3e4 FTFSSYAMS 1577SSISGSGGVTY 1801 CARGGNTYYYYYGM 1928 RASQSISSYLN 2094 AASSLQS 2110CQQSYSTPLTF 2174 YA DVW 009S-H08 LRP6e3e4 YTFTDYFMN 1673 GIINPSGDSTR1758 CARDDGLGGMDVW 1889 QASQDISNY 2076 AASSLQS 2110 CQQSYSTPLTF 2174 FALA 011S-E02 LRP6e3e4 YTFTYRYLH 1681 GGIIPIFGTAN 1749 CATDYGDYYYGMDVW1984 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA 009S-A09 LRP6e3e4YTFTYRYLH 1681 GRIIPILGSTNYA 1767 CTTDLWDYW 2048 QASQGITNY 2078 AASSLQS2110 CLQDYTDPFTF 2142 LN 011S-F02 LRP6e3e4 FTFSTYGMH 1584 SSISVSSGTTH1805 CARGGSGSYYYAFDIW 1930 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF2174 YA 011S-G02 LRP6e3e4 YTFTSYAMN 1678 GGIIPIFGTAN 1749 CARDASGGSTGWYY1887 RASQGISSYLA 2087 AASSLQS 2110 CQQAYSFPWTF 2150 YA FDSW 011S-H02LRP6e3e4 YTFTNNFMH 1676 GIINPSGGSTS 1761 CARGLYKRYSYGYG 1935 RASQSISSYLN2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA MDVW 009S-B09 LRP6e3e4 FSFNTYAMN1557 AVTSYDGGKK 1742 CARDAGGDYDYW 1885 QASQDISNY 2077 AASSLQS 2110CQQSYNTPRTF 2161 NYA LN 009S-C09 LRP6e3e4 GTFHTYGLS 1597 GGIIPIFGTAN1749 CARGSGWSGLDYW 1945 QASQDISNY 2077 DASNLET 2115 CQQSYTTPFTF 2180 YALN 011S-A03 LRP6e3e4 FTFSSYWMH 1580 STISGSGGRTY 1808 CATSPYGVFTLDYW 1994RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA 011S-B03 LRP6e3e4GTFSZYAIS 1604 GIINPSGGSTN 1760 CARAGYWSGYGYYG 1881 RASQSISSYLN 2094AASSLQS 2110 CQQSYSTPLTF 2174 YA MDVW 011S-C03 LRP6e3e4 YTFSYRYLH 1672GGIIPIFGTAN 1749 CASTVTTDAFDIW 1978 QASQDISNY 2077 DASSLES 2120CQQSYSFPPFTF 2168 YA LN 011S-D03 LRP6e3e4 FSFDDYGMS 1554 SVISSGGTIYYA1812 CARHLSSGYLSYYGM 1955 RASQSISSYLA 2093 AASTLQS 2113 CQQSYSTPLTF 2174DVW 009S-F09 LRP6e3e4 YSFTRTDMH 1670 GYISAYTGHTS 1778 CARDLGGTADYW 1900RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA 011S-E03 LRP6e3e4FTFSSYAMS 1577 SAISGSGGSTY 1786 CAKGGRDGYKGYFD 1861 KSSHSLLSTS 2070WASSRKS 2135 CQQYNNWPY 2186 YA YW TNRNQLA TF 009S-G09 LRP6e3e4 FTFSRHSMN1572 SYSSGNSGYT 1817 CARGDLEFDYW 1923 RASQGISNY 2083 SASSLQS 2132CQQGYNTPRTF 2154 NYA LA 011S-F03 LRP6e3e4 FTFSSYAMS 1577 SAISGSGGSTY1786 CAKGGRDGYKGYFD 1862 KSSQSVLYTT 2074 WASSRKS 2135 CQQYYSTPYTF 2189YA YW TNRNHIA 009S-H09 LRP6e3e4 FTFSSYAMS 1577 SAISGSGGSTY 1786CAKGGRDGYKGYFD 1863 KSSHSLLSTS 2069 WASSRKS 2135 CQQYYNIPYSF 2187 YA YWTNRNHLA 011S-G03 LRP6e3e4 YTFTYRYLH 1681 GRIIPIHGIANYA 1764CAREYSYGYFRYW 1922 RASQGISSYLA 2087 DASNLET 2115 CQQANSLFTF 2149009S-A10 LRP6e3e4 FTFTSSAMQ 1588 GIINPSGGSTIYA 1759 CASGDTYDLYSLDVW 1970RASQSISRW 2091 AASSLQS 2110 CQQAYSFPWTF 2150 LA 009S-B10 LRP6e3e4YIFTDYYMH 1669 GWINAGNGN 1772 CAKVASGWSWPFDIW 1871 RASQSISSYLN 2094AASSLQS 2110 CQQSYSTPLTF 2174 TTYA 011S-B04 LRP6e1e2 YTFTSYDIN 1679GIINPSGGSTS 1761 CTREHSYYYYGMDVW 2042 RASQSISSYLN 2094 AASSLQS 2110CQQSYSTPLTF 2174 YA 011S-C04 LRP6e1e2 GTFNSNAIS 1598 GWMNPNSG 1776CARDYYGSGSYNYG 1913 GASQSVPR 2066 GASQRAT 2124 CQQYHNWPP 2184 NTGYA MDVWNSLA EYTF 011S-D04 LRP6e1e2 YTFTSYDIN 1679 GIINPSGGSTS 1761CAREAYYYYYGMDVW 1916 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA011S-E04 LRP6e1e2 FTFSSZZMH 1581 SAIGTGGGTZYA 1782 CAKDLGRAAAGSMD 1850WASQSVRG 2106 DASNRAA 2117 CQHRSNWPLTF 2147 VW NYVA 011S-F04 LRP6e1e2YIFTDYYMH 1669 GRIIPILGRANYA 1765 CARGGYSTLDYW 1933 HGSQDISNY 2067DASNRQS 2119 CQQSFSTPRTF 2156 LN 011S-H04 LRP6e1e2 YIFTDYYMH 1669GRIIPILGRANYA 1765 CARGGYSTLDYW 1934 QASQDISNY 2077 AASTLQS 2113CQQSFSTPRTF 2156 LN 011S-A05 LRP6e1e2 FTFSSYAMH 1576 SAIGTGGGTYYA 1781CAKDLGRAAAGSMD 1848 WASQSVRG 2106 DASNRAG 2118 CQHRSNWPLTF 2147 VW NYVA011S-B05 LRP6e1e2 YZFTDYYMH 1683 GWMNPNSG 1776 CTRVAWGLDYW 2043RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 NTGYA 011S-C05 LRP6e1e2FTFSSYAMH 1576 SAIGTGGGTYYA 1781 CAKDLGRAAAGSMD 1849 WASQSVRG 2106DASNRAA 2117 CQHRSNWPLTF 2147 VW NYVA 1115.3 LRP6e1e2 GFSFSTS NLNGGSELAGYGTPFAY KASQSISYNLH YTSQSIS QQSNSWPLT  421.1 LRP6e1e2 GYTFTTYFPGNVNT EELQYYFDY SANSSVRFMF FTSNLAS QQYHSYPWT YW211.31. LRP6e3e4GFTFTSY SPYSGS RARPPIRLHPRGSVM RASQDVSTA SASFLYS QQSYTTPPT 57 DY VA

TABLE 2B Anti-LRP5/6 Antibody Clone IDs, Heavy Chain (HC) Seq ID Nos,and Binding Characteristics. Clone ID HC Seq ID NO Confirmed Binding001S-F11 66 LRP6e1e2 009S-G02 67 LRP6e1e2 009S-A03 68 LRP6e1e2 009S-D0369 LRP6e1e2 009S-F03 70 LRP6e1e2 009S-H03 71 LRP6e1e2 009S-A04 72LRP6e1e2 009S-B04 73 LRP6e3e4 009S-D04 74 LRP6e3e4 009S-E04 75 LRP6e3e4009S-F04 76 LRP6e3e4 009S-G04 77 LRP6e3e4 009S-H04 78 LRP6e3e4 009S-A0579 LRP6e3e4 013S-G04 80 LRP6e3e4 013S-H04 81 LRP6e3e4 013S-C05 82LRP6e3e4 013S-D05 83 LRP6e3e4 013S-G04 84 LRP6e3e4 013S-H04 85 LRP6e3e4013S-A05 86 LRP6e3e4 013S-C05 87 LRP6e3e4 013S-D05 88 LRP6e3e4 008S-D01133 LRP5

In certain embodiment, the LRP5/6 binding domain may be selected fromany binding domain that binds LRP5 or LRP6 with a K_(D) of less than orequal to about 1×10⁻⁴ M, less than or equal to about 1×10⁻⁵ M, less thanor equal to about 1×10⁻⁶ M, less than or equal to about 1×10⁻⁷ M, lessthan or equal to about 1×10⁻⁸ M, less than or equal to about 1×10⁻⁹ M,or less than or equal to about 1×10⁻¹⁰ M in the context of a Wntsurrogate molecule. In certain embodiment, the LRP5/6 binding domain maybe selected from any binding domain that binds LRP5 or LRP6 with a K_(D)of greater than or equal to about 1×10⁻⁴ M, greater than or equal toabout 1×10⁻⁵ M, greater than or equal to about 1×10⁻⁶ M, greater than orequal to about 1×10⁻⁷ M, greater than or equal to about 1×10⁻⁸ M,greater than or equal to about 1×10⁻⁹ M, or greater than about 1×10⁻¹⁰ Min the context of a Wnt surrogate molecule. In certain embodiment, theLRP5/6 binding domain may be selected from any binding domain that bindsLRP5 or LRP6 at high affinity, e.g. a K_(D) of less than about 1×10⁻⁷ M,less than about 1×10⁻⁸ M, less than about 1×10⁻⁹ M, or less than about1×10⁻¹⁰ M.

Other suitable LRP5/6 binding domains include, without limitation, denovo designed LRP5/6 binding proteins, antibody derived bindingproteins, e.g. scFv, Fab, etc. and other portions of antibodies thatspecifically bind to one or more Fzd proteins; VHH or sdAb derivedbinding domains; knottin-based engineered scaffolds; naturally occurringLRP5/6, including without limitation, DKK1, DKK2, DKK3, DKK4,sclerostin; Wise; fusions proteins comprising any of the above;derivatives of any of the above; variants of any of the above; andbiologically active fragments of any of the above, and the like. ALRP5/6 binding domain may be affinity selected to enhance binding.

Members of the Dickkopf (DKK) gene family (see Krupnik et al. (1999)Gene 238(2):301-13) include DKK-1, DKK-2, DKK-3, and DKK-4, and theDKK-3 related protein Soggy (Sgy). hDKKs 1-4 contain two distinctcysteine-rich domains in which the positions of 10 cysteine residues arehighly conserved between family members. Exemplary sequences of humanDkk genes and proteins are publicly available, e.g., Genbank accessionnumber NM_014419 (soggy-1); NM_014420 (DKK4); AF177394 (DKK-1); AF177395(DKK-2); NM_015881 (DKK3); and NM_014421 (DKK2). In some embodiments ofthe invention, the Lrp6 binding moiety is a DKK1 peptide, includingwithout limitation the C-terminal domain of human DKK1. The C-terminaldomain may comprise the sequence:

(SEQ ID NO: 2190) KMYHTKGQEGSVCLRSSDCASGLCCARHFWSKICKPVLKEGQVCTKHRRKGSHGLEIFQRCYCGEGLSCRIQKDHHQASNSSRLHTCQRH(see Genbank accession number NP_036374) or a biologically activefragment thereof.

Binding of DKK proteins to LRP5/6 are discussed, for example in Brottand Sokol Mol. Cell. Biol. 22 (17), 6100-6110 (2002); and Li et al. J.Biol. Chem. 277 (8), 5977-5981 (2002), each herein specificallyincorporated by reference. The corresponding region of human DKK2(Genbank reference NP_055236) may comprise the sequence:KMSHIKGHEGDPCLRSSDCIEGFCCARHFWTKICKPVLHQGEVCTKQRKKGSHGLEIFQRCDCAKGLSCKVWKDATYSSKARLHVCQK (SEQ ID NO:2191) or a biologicallyactive fragment thereof.

Antibodies that specifically bind to LRP5 or LRP6 are known in the artand are commercially available, or can be generated de novo. LRP5, LRP6or fragments thereof can be used as an immunogen or in screening assaysto develop an antibody. Examples of known antibodies include, withoutlimitation, those described in Gong et al. (2010) PLoS One. 5(9):e12682;Ettenberg et al. (2010) Proc Natl Acad Sci USA. 107(35):15473-8; andthose commercially available from, for example Santa Cruz biotechnologyantibody clone 1A12, which was raised against synthetic LRP5/6 of humanorigin and binds to both the full length and proteolytic fragment ofLRP6 and LRP5 of mouse and human origin; the monoclonal antibody 2611;Cell Signaling Technology antibody specific for LRP5 (D80F2), catalognumber 5731; etc.

In certain embodiments, Wnt surrogate molecules disclosed hereincomprise one or more polypeptides comprising two or more bindingregions. For example, the two or more binding regions may be two or moreFzd binding regions or two or more LRP5/6 binding regions, or they maycomprise one or more Fzd binding region and one or more LRP5/6 bindingregion. The binding regions may be directly joined or contiguous, or maybe separated by a linker, e.g. a polypeptide linker, or a non-peptidiclinker, etc. The length of the linker, and therefore the spacing betweenthe binding domains can be used to modulate the signal strength, and canbe selected depending on the desired use of the Wnt surrogate molecule.The enforced distance between binding domains can vary, but in certainembodiments may be less than about 100 angstroms, less than about 90angstroms, less than about 80 angstroms, less than about 70 angstroms,less than about 60 angstroms, or less than about 50 angstroms. In someembodiments the linker is a rigid linker, in other embodiments thelinker is a flexible linker. In certain embodiments where the linker isa peptide linker, it may be from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20 21, 22, 23, 24, 25, 26, 27, 28,29, 30 or more amino acids in length, and is of sufficient length andamino acid composition to enforce the distance between binding domains.In some embodiments, the linker comprises or consists of one or moreglycine and/or serine residues.

In particular embodiments, a Wnt surrogate molecule comprises apolypeptide sequence having at least 90%, at least 95%, at least 98% orat least 99% identity to a polypeptide sequence disclosed in any of SEQID NOs:89-128 or 134-157, or having at least 90%, at least 95%, at least98% or at least 99% identity to an antigen-binding fragment of apolypeptide sequence disclosed in any of SEQ ID NOs:89-128 or 134-157.In certain embodiments, the Wnt surrogate molecules comprises orconsists of a polypeptide sequence set forth in any of SEQ ID NOs:89-128or 134-157, or an antigen-binding fragment thereof. In particularembodiments, the antigen-binding fragment binds one or more Fzdreceptors and also binds LRP5 and/or LRP6.

Wnt surrogate molecule can be multimerized, e.g. through an Fc domain,by concatenation, coiled coils, polypeptide zippers, biotin/avidin orstreptavidin multimerization, and the like. The Wnt surrogate moleculescan also be joined to a moiety such as PEG, Fc, etc., as known in theart to enhance stability in vivo.

In certain embodiments, a Wnt surrogate molecule directly activatescanonical Wnt signaling through binding to one or more Fzd proteins andto LRP5/6, particularly by binding to these proteins on a cell surface,e.g., the surface of a human cell. The direct activation of Wntsignaling by a Wnt surrogate molecule is in contrast to potentiation ofWrit signaling, which enhances activity only when native Wnt proteinsare present.

Wnt surrogate molecules may activate Wnt signaling, e.g., by mimickingthe effect or activity of a Wnt protein binding to a frizzled protein.The ability of the Wnt surrogate molecules of the invention to mimic theactivity of Wnt can be confirmed by a number of assays. The Wntsurrogate molecules typically initiate a reaction or activity that issimilar to or the same as that initiated by the receptor's naturalligand. In particular, the Wnt surrogate molecules of the inventionenhance the canonical Wnt/β-catenin signaling pathway. As used herein,the term “enhances” refers to a measurable increase in the level ofWnt/β-catenin signaling compared with the level in the absence of a Wntsurrogate molecule of the invention.

Various methods are known in the art for measuring the level ofcanonical Wnt/β-catenin signaling. These include, but are not limited toassays that measure: Wnt/β-catenin target gene expression; TCF reportergene expression; β-catenin stabilization; LRP phosphorylation; Axintranslocation from cytoplasm to cell membrane and binding to LRP. Thecanonical Wnt/β-catenin signaling pathway ultimately leads to changes ingene expression through the transcription factors TCF7, TCF7L1, TCF7L2(a.k.a., TCF4), and LEF. The transcriptional response to Wnt activationhas been characterized in a number of cells and tissues. As such, globaltranscriptional profiling by methods well known in the art can be usedto assess Wnt/β-catenin signaling activation or inhibition.

Changes in Wnt-responsive gene expression are generally mediated by TCFand LEF transcription factors. A TCF reporter assay assesses changes inthe transcription of TCF/LEF controlled genes to determine the level ofWnt/β-catenin signaling. A TCF reporter assay was first described byKorinek, V. et al., 1997. Also known as TOP/FOP this method involves theuse of three copies of the optimal TCF motif CCTTTGATC, or three copiesof the mutant motif CCTTTGGCC, upstream of a minimal c-Fos promoterdriving luciferase expression (pTOPFI_ASH and pFOPFI_ASH, respectively)to determine the transactivational activity of endogenousp-catenin/TCF4. A higher ratio of these two reporter activities(TOP/FOP) indicates higher β-catenin/TCF4 activity, whereas a lowerratio of these two reporter activities indicates lower β-catenin/TCF4activity.

Various other reporter transgenes that respond to Wnt signals existintact in animals and therefore, effectively reflect endogenous Wntsignaling. These reporters are based on a multimerized TCF binding site,which drives expression of LacZ or GFP, which are readily detectable bymethods known in the art. These reporter genes include: TOP-GAL,BAT-GAL, ins-TOPE FP, ins-TOPGAL, LEF-EGFP, Axin2-LacZ, Axin2-d2EGFP,Lgr5tm1 (cre/ERT2), TOPdGFP.

The recruitment of dephosphorylated β-catenin to the membrane,stabilization and phosphorylation status of β-catenin, and translocationof β-catenin to the nucleus (Klapholz-Brown Z et al., PLoS One. 2(9)e945, 2007), in some cases mediated by complex formation with TCFtranscription factors and TNIK are key steps in the Writ signalingpathway. Stabilization is mediated by Disheveled family proteins thatinhibit the “destruction” complex so that degradation of intracellularβ-catenin is reduced, and translocation of β-catenin to the nucleusfollows thereafter. Therefore, measuring the level and location ofβ-catenin in a cell is a good reflection of the level of Wnt/β,-cateninsignaling. A non-limiting example of such an assay is the “BioImageβ-Catenin Redistribution Assay” (Thermo Scientific) which providesrecombinant U20S cells that stably express human β-catenin fused to theC-terminus of enhanced green fluorescent protein (EGFP). Imaging andanalysis is performed with a fluorescence microscope or HCS platformallowing the levels and distribution of EGFP-β-catenin to be visualized.

Another way, in which the destruction complex is inhibited, is byremoval of Axin by recruitment of Axin to the cytoplasmic tail of theWnt co-receptor LRP. Axin has been shown to bind preferentially to aphosphorylated form of the LRP tail. Visualization of Axintranslocation, for example with a GFP-Axin fusion protein, is thereforeanother method for assessing levels of Wnt/β-catenin signaling.

In certain embodiments, a Wnt surrogate molecule enhances or increasescanonical Wnt pathway signaling, e.g., β-catenin signaling, by at least30%, 35%, 40%, 45%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%,150%, 200%, 250%, 300%, 400% or 500%, as compared to the β-cateninsignaling induced by a neutral substance or negative control as measuredin an assay described above, for example as measured in the TOPFlashassay. A negative control may be included in these assays. In particularembodiments, Wnt surrogate molecules may enhance β-catenin signaling bya factor of 2×, 5×, 10×, 100×, 1000×, 10000× or more as compared to theactivity in the absence of the Wnt surrogate molecule when measured inan assay described above, for example when measured in the TOPFlashassay, or any of the other assays mentioned herein.

“Wnt gene product” or “Wnt polypeptide” when used herein encompassnative sequence Writ polypeptides, Wnt polypeptide variants, Wntpolypeptide fragments and chimeric Wnt polypeptides. In particularembodiments, a Wnt polypeptide is a native human full length mature Wntprotein.

For example, human native sequence Wnt proteins of interest in thepresent application include the following: Wnt-1 (GenBank Accession No.NM_005430); Wnt-2 (GenBank Accession No. NM_003391); Wnt-2B (Wnt-13)(GenBank Accession No. NM_004185 (isoform 1), NM_024494.2 (isoform 2)),Wnt-3 (RefSeq.: NM_030753), Wnt3a (GenBank Accession No. NM_033131),Wnt-4 (GenBank Accession No. NM_030761), Wnt-5A (GenBank Accession No.NM_003392), Wnt-5B (GenBank Accession No. NM_032642), Wnt-6 (GenBankAccession No. NM_006522), Wnt-7A (GenBank Accession No. NM_004625),Wnt-7B (GenBank Accession No. NM_058238), Wnt-8A (GenBank Accession No.NM_058244), Wnt-8B (GenBank Accession No. NM_003393), Wnt-9A (Wnt-14)(GenBank Accession No. NM_003395), Wnt-9B (Wnt-15) (GenBank AccessionNo. NM_003396), Wnt-1 OA (GenBank Accession No. NM_025216), Wnt-10B(GenBank Accession No. NM_003394), Wnt-11 (GenBank Accession No.NM_004626), Wnt-16 (GenBank Accession No. NM_016087)). Although eachmember has varying degrees of sequence identity with the family, allencode small (i.e., 39-46 kD), acylated, palmitoylated, secretedglycoproteins that contain 23-24 conserved cysteine residues whosespacing is highly conserved (McMahon, A P et al., Trends Genet. 1992; 8:236-242; Miller, J R. Genome Biol. 2002; 3(1): 3001.1-3001.15). Othernative sequence Wnt polypeptides of interest include orthologs of theabove from any mammal, including domestic and farm animals, and zoo,laboratory or pet animals, such as dogs, cats, cattle, horses, sheep,pigs, goats, rabbits, rats, mice, frogs, zebra fish, fruit fly, worm,etc.

“Wnt pathway signaling” or “Wnt signaling” is used herein to refer tothe mechanism by which a biologically active Wnt exerts its effects upona cell to modulate a cell's activity. Wnt proteins modulate cellactivity by binding to Wnt receptors, including proteins from theFrizzled (Fzd) family of proteins, proteins from the ROR family ofproteins, the proteins LRP5, LRP6 from the LRP family of proteins, theprotein FRL1/crypto, and the protein Derailed/Ryk. Once activated by Wntbinding, the Wnt receptor(s) will activate one or more intracellularsignaling cascades. These include the canonical Wnt signaling pathway;the Wnt/planar cell polarity (Wnt/PCP) pathway; the Wnt-calcium(Wnt/Ca²⁺) pathway (Giles, R H et al. (2003) Biochim Biophys Acta 1653,1-24; Peifer, M. et al. (1994) Development 120: 369-380; Papkoff, J. etal (1996) Mol. Cell Biol. 16: 2128-2134; Veeman, M. T. et al. (2003)Dev. Cell 5: 367-377); and other Writ signaling pathways as is wellknown in the art.

For example, activation of the canonical Wnt signaling pathway resultsin the inhibition of phosphorylation of the intracellular proteinβ-catenin, leading to an accumulation of β-catenin in the cytosol andits subsequent translocation to the nucleus where it interacts withtranscription factors, e.g. TCF/LEF, to activate target genes.Activation of the Wnt/PCP pathway activates RhoA, c-Jun N-terminalkinase (JNK), and nemo-like kinase (NLK) signaling cascades to controlsuch biological processes as tissue polarity and cell movement.Activation of the Wnt/Ca²⁺ by, for example, binding of Wnt-4, Wnt-5A orWnt-11, elicits an intracellular release of calcium ions, whichactivates calcium sensitive enzymes like protein kinase C (PKC),calcium-calmodulin dependent kinase II (CamKII) or calcineurin (CaCN).By assaying for activity of the above signaling pathways, the biologicalactivity of an antibody or antigen-binding fragment thereof, e.g., a Wntsurrogate molecule, can be readily determined.

In certain embodiments, functional properties of Wnt surrogate moleculesmay be assessed using a variety of methods known to the skilled person,including e.g., affinity/binding assays (for example, surface plasmonresonance, competitive inhibition assays), cytotoxicity assays, cellviability assays, cell proliferation or differentiation assays inresponse to a Wnt, cancer cell and/or tumor growth inhibition using invitro or in vivo models, including but not limited to any describedherein. The Wnt surrogate molecules described herein may also be testedfor effects on Fzd receptor internalization, in vitro and in vivoefficacy, etc. Such assays may be performed using well-establishedprotocols known to the skilled person (see e.g., Current Protocols inMolecular Biology (Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc.,NY, NY); Current Protocols in Immunology (Edited by: John E. Coligan,Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober2001 John Wiley & Sons, NY, NY); or commercially available kits.

In certain embodiments, a Fzd-binding region of a Wnt surrogate molecule(e.g., an antigen-binding fragment of an anti-Fzd antibody) comprisesone or more of the CDRs of the anti-Fzd antibodies described herein. Incertain embodiments, a LRP5/6-binding region of a Wnt surrogate molecule(e.g., an antigen-binding fragment of an anti-LRP5/6 antibody) comprisesone or more of the CDRs of the anti-LRP5/6 antibodies described herein.In this regard, it has been shown in some cases that the transfer ofonly the VHCDR3 of an antibody can be performed while still retainingdesired specific binding (Barbas et al., PNAS (1995) 92: 2529-2533). Seealso, McLane et al., PNAS (1995) 92:5214-5218, Barbas et al., J. Am.Chem. Soc. (1994) 116:2161-2162.

Also disclosed herein is a method for obtaining an antibody or antigenbinding domain specific for a Fzd receptor, the method comprisingproviding by way of addition, deletion, substitution or insertion of oneor more amino acids in the amino acid sequence of a VH domain set outherein or a VH domain which is an amino acid sequence variant of the VHdomain, optionally combining the VH domain thus provided with one ormore VL domains, and testing the VH domain or VH/VL combination orcombinations to identify a specific binding member or an antibodyantigen binding domain specific for one or more Fzd receptor andoptionally with one or more desired properties. The VL domains may havean amino acid sequence which is substantially as set out herein. Ananalogous method may be employed in which one or more sequence variantsof a VL domain disclosed herein are combined with one or more VHdomains.

In particular embodiments, Wnt surrogate molecules are water soluble. By“water soluble” it is meant a composition that is soluble in aqueousbuffers in the absence of detergent, usually soluble at a concentrationthat provides a biologically effective dose of the polypeptide.Compositions that are water soluble form a substantially homogenouscomposition that has a specific activity that is at least about 5% thatof the starting material from which it was purified, usually at leastabout 10%, 20%, or 30% that of the starting material, more usually about40%, 50%, or 60% that of the starting material, and may be about 50%,about 90% or greater. Wnt surrogate molecules disclosed herein typicallyform a substantially homogeneous aqueous solution at concentrations ofat least 25 μM and higher, e.g. at least 25 μM, 40 μM, or 50 μM, usuallyat least 60 μM, 70 μM, 80 μM, or 90 μM, sometimes as much as 100 μM, 120μM, or 150 μM. In other words, Wnt surrogate molecules disclosed hereintypically form a substantially homogeneous aqueous solution atconcentrations of about 0.1 mg/ml, about 0.5 mg/ml, of about 1 mg/ml ormore.

An antigen or epitope that “specifically binds” or “preferentiallybinds” (used interchangeably herein) to an antibody or antigen-bindingfragment thereof is a term well understood in the art, and methods todetermine such specific or preferential binding are also well known inthe art. A molecule, e.g., a Wnt surrogate molecule, is said to exhibit“specific binding” or “preferential binding” if it reacts or associatesmore frequently, more rapidly, with greater duration and/or with greateraffinity with a particular cell or substance than it does withalternative cells or substances. A molecule or binding region thereof,e.g., a Wnt surrogate molecule or binding region thereof, “specificallybinds” or “preferentially binds” to a target antigen, e.g., a Fzdreceptor, if it binds with greater affinity, avidity, more readily,and/or with greater duration than it binds to other substances. Forexample, a Wnt surrogate molecule or binding region thereof thatspecifically or preferentially binds to the Fzd1 receptor is an antibodythat binds to the Fzd1 receptor with greater affinity, avidity, morereadily, and/or with greater duration than it binds to other Fzdreceptors or non-Fzd proteins. It is also understood by reading thisdefinition that, for example, a Wnt surrogate molecule or binding regionthereof that specifically or preferentially binds to a first target mayor may not specifically or preferentially bind to a second target. Assuch, “specific binding” or “preferential binding” does not necessarilyrequire (although it can include) exclusive binding. Generally, but notnecessarily, reference to binding means preferential binding.

In some embodiments, any of the one or more Fzd binding region of a Wntsurrogate molecule binds to one, two, three, four, five or moredifferent frizzled proteins, e.g., one or more of human frizzledproteins Fzd1, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8, Fzd9, Fzd10. Insome embodiments, any of the Fzd binding regions binds to Fzd1, Fzd2,Fzd5, Fzd7 and Fzd8. In various embodiments, any of the Fzd bindingregions binds to: (i) Fzd1, Fzd2, Fzd7 and Fzd9; (ii) Fzd1, Fzd2 andFzd7; (iii) Fzd5 and Fzd8; (iv) Fzd5, Fzd7 and Fzd8; (v) Fzd1, Fzd4,Fzd5 and Fzd8; (vi) Fzd1, Fzd2, Fzd5, Fzd7 and Fzd8; (vii) Fzd4 andFzd9; (viii) Fzd9 and Fzd10; (ix) Fzd5, Fzd8 and Fzd10; or (x) Fzd4,Fzd5 and Fzd8; Fzd1, Fzd5, Fzd7 and Fzd8. In some embodiments, the Fzdbinding region is selective for one or more Fzd protein of interest,e.g. having a specificity for the one or more desired Fzd protein of atleast 10-fold, 25-fold, 50-fold, 100-fold, 200-fold or more relative toother Fzd proteins. In some embodiments, any of the one or more Fzdbinding region of a Wnt surrogate molecule is monospecific and binds orspecifically binds to only one of Fzd1, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6,Fzd7, Fzd8, Fzd9, or Fzd10.

In some embodiments, any of the one or more LRP5/6 binding region of aWnt surrogate molecule binds to one or both of LRP5/6. For convenience,the term “LRP5/6” is used to refer collectively to either or both ofLRP5 and/or LRP6.

Immunological binding generally refers to the non-covalent interactionsof the type which occur between an immunoglobulin molecule and anantigen for which the immunoglobulin is specific, for example by way ofillustration and not limitation, as a result of electrostatic, ionic,hydrophilic and/or hydrophobic attractions or repulsion, steric forces,hydrogen bonding, van der Waals forces, and other interactions. Thestrength, or affinity of immunological binding interactions can beexpressed in terms of the dissociation constant (K_(d)) of theinteraction, wherein a smaller K_(d) represents a greater affinity.Immunological binding properties of selected polypeptides can bequantified using methods well known in the art. One such method entailsmeasuring the rates of antigen-binding site/antigen complex formationand dissociation, wherein those rates depend on the concentrations ofthe complex partners, the affinity of the interaction, and on geometricparameters that equally influence the rate in both directions. Thus,both the “on rate constant” (K_(on)) and the “off rate constant”(K_(off)) can be determined by calculation of the concentrations and theactual rates of association and dissociation. The ratio ofK_(off)/K_(on) enables cancellation of all parameters not related toaffinity, and is thus equal to the dissociation constant K_(d). See,generally, Davies et al. (1990) Annual Rev. Biochem. 59:439-473.

In certain embodiments, the Wnt surrogate molecules or binding regionsthereof described herein have an affinity of less than about 10,000,less than about 1000, less than about 100, less than about 10, less thanabout 1 or less than about 0.1 nM, and in some embodiments, theantibodies may have even higher affinity for one or more Fzd receptor orLRP5 or LRP6 receptor.

The constant regions of immunoglobulins show less sequence diversitythan the variable regions, and are responsible for binding a number ofnatural proteins to elicit important biochemical events. In humans,there are five different classes of antibodies including IgA (whichincludes subclasses IgA1 and IgA2), IgD, IgE, IgG (which includessubclasses IgG1, IgG2, IgG3, and IgG4), and IgM. The distinguishingfeatures between these antibody classes are their constant regions,although subtler differences may exist in the V region.

The Fc region of an antibody interacts with a number of Fc receptors andligands, imparting an array of important functional capabilitiesreferred to as effector functions. For IgG, the Fc region comprises Igdomains CH2 and CH3 and the N-terminal hinge leading into CH2. Animportant family of Fc receptors for the IgG class are the Fc gammareceptors (FcγRs). These receptors mediate communication betweenantibodies and the cellular arm of the immune system (Raghavan et al.,1996, Annu Rev Cell Dev Biol 12:181-220; Ravetch et al., 2001, Annu RevImmunol 19:275-290). In humans this protein family includes FcγRI(CD64), including isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32),including isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb(including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16),including isoforms FcγRIIIa (including allotypes V158 and F158) andFcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2) (Jefferiset al., 2002, Immunol Lett 82:57-65). These receptors typically have anextracellular domain that mediates binding to Fc, a membrane spanningregion, and an intracellular domain that may mediate some signalingevent within the cell. These receptors are expressed in a variety ofimmune cells including monocytes, macrophages, neutrophils, dendriticcells, eosinophils, mast cells, platelets, B cells, large granularlymphocytes, Langerhans' cells, natural killer (NK) cells, and T cells.Formation of the Fc/FcγR complex recruits these effector cells to sitesof bound antigen, typically resulting in signaling events within thecells and important subsequent immune responses such as release ofinflammation mediators, B cell activation, endocytosis, phagocytosis,and cytotoxic attack.

The ability to mediate cytotoxic and phagocytic effector functions is apotential mechanism by which antibodies destroy targeted cells. Thecell-mediated reaction wherein nonspecific cytotoxic cells that expressFcγRs recognize bound antibody on a target cell and subsequently causelysis of the target cell is referred to as antibody dependentcell-mediated cytotoxicity (ADCC) (Raghavan et al., 1996, Annu Rev CellDev Biol 12:181-220; Ghetie et al., 2000, Annu Rev Immunol 18:739-766;Ravetch et al., 2001, Annu Rev Immunol 19:275-290). The cell-mediatedreaction wherein nonspecific cytotoxic cells that express FcγRsrecognize bound antibody on a target cell and subsequently causephagocytosis of the target cell is referred to as antibody dependentcell-mediated phagocytosis (ADCP). All FcγRs bind the same region on Fc,at the N-terminal end of the Cg2 (CH2) domain and the preceding hinge.This interaction is well characterized structurally (Sondermann et al.,2001, J Mol Biol 309:737-749), and several structures of the human Fcbound to the extracellular domain of human FcγRIIIb have been solved(pdb accession code 1E4K) (Sondermann et al., 2000, Nature 406:267-273.)(pdb accession codes 11 IIS and 11 IX) (Radaev et al., 2001, J Biol Chem276:16469-16477.)

The different IgG subclasses have different affinities for the FcγRs,with IgG1 and IgG3 typically binding substantially better to thereceptors than IgG2 and IgG4 (Jefferis et al., 2002, Immunol Lett82:57-65). All FcγRs bind the same region on IgG Fc, yet with differentaffinities: the high affinity binder FcγRI has a K_(d) for IgG1 of 10⁻⁸M⁻¹, whereas the low affinity receptors FcγRII and FcγRIII generallybind at 10⁻⁶ and 10⁻⁵ respectively. The extracellular domains ofFcγRIIIa and FcγRIIIb are 96% identical; however, FcγRIIIb does not havean intracellular signaling domain. Furthermore, whereas FcγRI,FcγRIIa/c, and FcγRIIIa are positive regulators of immunecomplex-triggered activation, characterized by having an intracellulardomain that has an immunoreceptor tyrosine-based activation motif(ITAM), FcγRIIb has an immunoreceptor tyrosine-based inhibition motif(ITIM) and is therefore inhibitory. Thus the former are referred to asactivation receptors, and FcγRIIb is referred to as an inhibitoryreceptor. The receptors also differ in expression pattern and levels ondifferent immune cells. Yet another level of complexity is the existenceof a number of FcγR polymorphisms in the human proteome. A particularlyrelevant polymorphism with clinical significance is V158/F158 FcγRIIIa.Human IgG1 binds with greater affinity to the V158 allotype than to theF158 allotype. This difference in affinity, and presumably its effect onADCC and/or ADCP, has been shown to be a significant determinant of theefficacy of the anti-CD20 antibody rituximab (Rituxan®, a registeredtrademark of IDEC Pharmaceuticals Corporation). Subjects with the V158allotype respond favorably to rituximab treatment; however, subjectswith the lower affinity F158 allotype respond poorly (Cartron et al.,2002, Blood 99:754-758). Approximately 10-20% of humans are V158/V158homozygous, 45% are V158/F158 heterozygous, and 35-45% of humans areF158/F158 homozygous (Lehrbecher et al., 1999, Blood 94:4220-4232;Cartron et al., 2002, Blood 99:754-758). Thus 80-90% of humans are poorresponders, that is, they have at least one allele of the F158 FcγRIIIa.

The Fc region is also involved in activation of the complement cascade.In the classical complement pathway, C1 binds with its C1q subunits toFc fragments of IgG or IgM, which has formed a complex with antigen(s).In certain embodiments of the invention, modifications to the Fc regioncomprise modifications that alter (either enhance or decrease) theability of a Fzd-specific antibody as described herein to activate thecomplement system (see e.g., U.S. Pat. No. 7,740,847). To assesscomplement activation, a complement-dependent cytotoxicity (CDC) assaymay be performed (See, e.g., Gazzano-Santoro et al., J. Immunol.Methods, 202:163 (1996)).

Thus in certain embodiments, the present invention provides anti-Fzdantibodies having a modified Fc region with altered functionalproperties, such as reduced or enhanced CDC, ADCC, or ADCP activity, orenhanced binding affinity for a specific FcγR or increased serumhalf-life. Other modified Fc regions contemplated herein are described,for example, in issued U.S. Pat. Nos. 7,317,091; 7,657,380; 7,662,925;6,538,124; 6,528,624; 7,297,775; 7,364,731; Published U.S. ApplicationsUS2009092599; US20080131435; US20080138344; and published InternationalApplications WO2006/105338; WO2004/063351; WO2006/088494; WO2007/024249.

In certain embodiments, Wnt surrogate molecules comprise antibodyvariable domains with the desired binding specificities fused toimmunoglobulin constant domain sequences. In certain embodiments, thefusion is with an Ig heavy chain constant domain, comprising at leastpart of the hinge, C_(H)2, and C_(H)3 regions. In particularembodiments, the first heavy-chain constant region (C_(H)1) containingthe site necessary for light chain bonding, present in at least one ofthe fusions. DNAs encoding the immunoglobulin heavy chain fusions and,if desired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable host cell.This provides for greater flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yield of the desired bispecific antibody. It is,however, possible to insert the coding sequences for two or all threepolypeptide chains into a single expression vector when the expressionof at least two polypeptide chains in equal ratios results in highyields or when the ratios have no significant effect on the yield of thedesired chain combination.

Wnt surrogate molecules disclosed herein may also be modified to includean epitope tag or label, e.g., for use in purification or diagnosticapplications. There are many linking groups known in the art for makingantibody conjugates, including, for example, those disclosed in U.S.Pat. No. 5,208,020 or EP Patent 0 425 235 B1, and Chari et al., CancerResearch 52: 127-131 (1992). The linking groups include disulfidegroups, thioether groups, acid labile groups, photolabile groups,peptidase labile groups, or esterase labile groups, as disclosed in theabove-identified patents, disulfide and thioether groups beingpreferred.

In certain embodiments, anti-LRP5/6 antibodies and antigen-bindingfragments thereof and/or anti-Fzd antibodies and antigen-bindingfragments thereof present within a Wnt surrogate molecule aremonoclonal. In certain embodiments, they are humanized.

The present invention further provides in certain embodiments anisolated nucleic acid encoding a polypeptide present in a Wnt surrogatemolecule disclosed herein. Nucleic acids include DNA and RNA. These andrelated embodiments may include polynucleotides encoding antibodyfragments that bind one or more Fzd receptors and/or LRP5 or LRP6 asdescribed herein. The term “isolated polynucleotide” as used hereinshall mean a polynucleotide of genomic, cDNA, or synthetic origin, orsome combination thereof, which by virtue of its origin, the isolatedpolynucleotide: (1) is not associated with all or a portion of apolynucleotide in which the isolated polynucleotide is found in nature;(2) is linked to a polynucleotide to which it is not linked in nature,or (3) does not occur in nature as part of a larger sequence. Anisolated polynucleotide may include naturally occurring and/orartificial sequences.

The term “operably linked” means that the components to which the termis applied are in a relationship that allows them to carry out theirinherent functions under suitable conditions. For example, atranscription control sequence “operably linked” to a protein codingsequence is ligated thereto so that expression of the protein codingsequence is achieved under conditions compatible with thetranscriptional activity of the control sequences.

The term “control sequence” as used herein refers to polynucleotidesequences that can affect expression, processing or intracellularlocalization of coding sequences to which they are ligated or operablylinked. The nature of such control sequences may depend upon the hostorganism. In particular embodiments, transcription control sequences forprokaryotes may include a promoter, ribosomal binding site, andtranscription termination sequence. In other particular embodiments,transcription control sequences for eukaryotes may include promoterscomprising one or a plurality of recognition sites for transcriptionfactors, transcription enhancer sequences, transcription terminationsequences and polyadenylation sequences. In certain embodiments,“control sequences” can include leader sequences and/or fusion partnersequences.

The term “polynucleotide” as referred to herein means single-stranded ordouble-stranded nucleic acid polymers. In certain embodiments, thenucleotides comprising the polynucleotide can be ribonucleotides ordeoxyribonucleotides or a modified form of either type of nucleotide.Said modifications include base modifications such as bromouridine,ribose modifications such as arabinoside and 2′,3′-dideoxyribose andinternucleotide linkage modifications such as phosphorothioate,phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,phosphoroanilothioate, phoshoraniladate and phosphoroamidate. The term“polynucleotide” specifically includes single and double stranded formsof DNA.

The term “naturally occurring nucleotides” includes deoxyribonucleotidesand ribonucleotides. The term “modified nucleotides” includesnucleotides with modified or substituted sugar groups and the like. Theterm “oligonucleotide linkages” includes oligonucleotide linkages suchas phosphorothioate, phosphorodithioate, phosphoroselenoate,phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate,phosphoroamidate, and the like. See, e.g., LaPlanche et al., 1986, Nucl.Acids Res., 14:9081; Stec et al., 1984, J. Am. Chem. Soc., 106:6077;Stein et al., 1988, Nucl. Acids Res., 16:3209; Zon et al., 1991,Anti-Cancer Drug Design, 6:539; Zon et al., 1991, OLIGONUCLEOTIDES ANDANALOGUES: A PRACTICAL APPROACH, pp. 87-108 (F. Eckstein, Ed.), OxfordUniversity Press, Oxford England; Stec et al., U.S. Pat. No. 5,151,510;Uhlmann and Peyman, 1990, Chemical Reviews, 90:543, the disclosures ofwhich are hereby incorporated by reference for any purpose. Anoligonucleotide can include a detectable label to enable detection ofthe oligonucleotide or hybridization thereof.

The term “vector” is used to refer to any molecule (e.g., nucleic acid,plasmid, or virus) used to transfer coding information to a host cell.The term “expression vector” refers to a vector that is suitable fortransformation of a host cell and contains nucleic acid sequences thatdirect and/or control expression of inserted heterologous nucleic acidsequences. Expression includes, but is not limited to, processes such astranscription, translation, and RNA splicing, if introns are present.

As will be understood by those skilled in the art, polynucleotides mayinclude genomic sequences, extra-genomic and plasmid-encoded sequencesand smaller engineered gene segments that express, or may be adapted toexpress, proteins, polypeptides, peptides and the like. Such segmentsmay be naturally isolated, or modified synthetically by the skilledperson.

As will be also recognized by the skilled artisan, polynucleotides maybe single-stranded (coding or antisense) or double-stranded, and may beDNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules mayinclude HnRNA molecules, which contain introns and correspond to a DNAmolecule in a one-to-one manner, and mRNA molecules, which do notcontain introns. Additional coding or non-coding sequences may, but neednot, be present within a polynucleotide according to the presentdisclosure, and a polynucleotide may, but need not, be linked to othermolecules and/or support materials. Polynucleotides may comprise anative sequence or may comprise a sequence that encodes a variant orderivative of such a sequence.

It will be appreciated by those of ordinary skill in the art that, as aresult of the degeneracy of the genetic code, there are many nucleotidesequences that encodes an antibody as described herein. Some of thesepolynucleotides bear minimal sequence identity to the nucleotidesequence of the native or original polynucleotide sequence encoding apolypeptide within a Wnt surrogate molecule. Nonetheless,polynucleotides that vary due to differences in codon usage areexpressly contemplated by the present disclosure. In certainembodiments, sequences that have been codon-optimized for mammalianexpression are specifically contemplated.

Therefore, in another embodiment of the invention, a mutagenesisapproach, such as site-specific mutagenesis, may be employed for thepreparation of variants and/or derivatives of the polypeptides describedherein. By this approach, specific modifications in a polypeptidesequence can be made through mutagenesis of the underlyingpolynucleotides that encode them. These techniques provide astraightforward approach to prepare and test sequence variants, forexample, incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into thepolynucleotide.

Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Mutations may be employed in aselected polynucleotide sequence to improve, alter, decrease, modify, orotherwise change the properties of the polynucleotide itself, and/oralter the properties, activity, composition, stability, or primarysequence of the encoded polypeptide.

In certain embodiments, the inventors contemplate the mutagenesis of thepolynucleotide sequences that encode a polypeptide present in a Wntsurrogate molecule, to alter one or more properties of the encodedpolypeptide, such as the binding affinity, or the function of aparticular Fc region, or the affinity of the Fc region for a particularFcγR. The techniques of site-specific mutagenesis are well-known in theart, and are widely used to create variants of both polypeptides andpolynucleotides. For example, site-specific mutagenesis is often used toalter a specific portion of a DNA molecule. In such embodiments, aprimer comprising typically about 14 to about 25 nucleotides or so inlength is employed, with about 5 to about 10 residues on both sides ofthe junction of the sequence being altered.

As will be appreciated by those of skill in the art, site-specificmutagenesis techniques have often employed a phage vector that exists inboth a single stranded and double stranded form. Typical vectors usefulin site-directed mutagenesis include vectors such as the M13 phage.These phages are readily commercially available and their use isgenerally well-known to those skilled in the art. Double-strandedplasmids are also routinely employed in site directed mutagenesis thateliminates the step of transferring the gene of interest from a plasmidto a phage.

The preparation of sequence variants of the selected peptide-encodingDNA segments using site-directed mutagenesis provides a means ofproducing potentially useful species and is not meant to be limiting asthere are other ways in which sequence variants of peptides and the DNAsequences encoding them may be obtained. For example, recombinantvectors encoding the desired peptide sequence may be treated withmutagenic agents, such as hydroxylamine, to obtain sequence variants.Specific details regarding these methods and protocols are found in theteachings of Maloy et al., 1994; Segal, 1976; Prokop and Bajpai, 1991;Kuby, 1994; and Maniatis et al., 1982, each incorporated herein byreference, for that purpose.

In many embodiments, one or more nucleic acids encoding a polypeptide ofa Wnt surrogate molecule are introduced directly into a host cell, andthe cell incubated under conditions sufficient to induce expression ofthe encoded polypeptides. The Wnt surrogate polypeptides of thisdisclosure may be prepared using standard techniques well known to thoseof skill in the art in combination with the polypeptide and nucleic acidsequences provided herein. The polypeptide sequences may be used todetermine appropriate nucleic acid sequences encoding the particularpolypeptide disclosed thereby. The nucleic acid sequence may beoptimized to reflect particular codon “preferences” for variousexpression systems according to standard methods well known to those ofskill in the art.

According to certain related embodiments there is provided a recombinanthost cell which comprises one or more constructs as described herein,e.g., a vector comprising a nucleic acid encoding a Wnt surrogatemolecule or polypeptide thereof; and a method of production of theencoded product, which method comprises expression from encoding nucleicacid therefor. Expression may conveniently be achieved by culturingunder appropriate conditions recombinant host cells containing thenucleic acid. Following production by expression, an antibody orantigen-binding fragment thereof, may be isolated and/or purified usingany suitable technique, and then used as desired.

Polypeptides, and encoding nucleic acid molecules and vectors, may beisolated and/or purified, e.g. from their natural environment, insubstantially pure or homogeneous form, or, in the case of nucleic acid,free or substantially free of nucleic acid or genes of origin other thanthe sequence encoding a polypeptide with the desired function. Nucleicacid may comprise DNA or RNA and may be wholly or partially synthetic.Reference to a nucleotide sequence as set out herein encompasses a DNAmolecule with the specified sequence, and encompasses a RNA moleculewith the specified sequence in which U is substituted for T, unlesscontext requires otherwise.

Systems for cloning and expression of a polypeptide in a variety ofdifferent host cells are well known. Suitable host cells includebacteria, mammalian cells, yeast and baculovirus systems. Mammalian celllines available in the art for expression of a heterologous polypeptideinclude Chinese hamster ovary cells, HeLa cells, baby hamster kidneycells, NSO mouse melanoma cells and many others. A common, preferredbacterial host is E. coli.

The expression of polypeptides, e.g., antibodies and antigen-bindingfragments thereof, in prokaryotic cells such as E. coli is wellestablished in the art. For a review, see for example Pluckthun, A.Bio/Technology 9: 545-551 (1991). Expression in eukaryotic cells inculture is also available to those skilled in the art as an option forproduction of antibodies or antigen-binding fragments thereof, seerecent reviews, for example Ref, M. E. (1993) Curr. Opinion Biotech. 4:573-576; Trill J. J. et al. (1995) Curr. Opinion Biotech 6: 553-560.

Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminatorsequences, polyadenylation sequences, enhancer sequences, marker genesand other sequences as appropriate. Vectors may be plasmids, viral e.g.phage, or phagemid, as appropriate. For further details see, forexample, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrooket al., 1989, Cold Spring Harbor Laboratory Press. Many known techniquesand protocols for manipulation of nucleic acid, for example inpreparation of nucleic acid constructs, mutagenesis, sequencing,introduction of DNA into cells and gene expression, and analysis ofproteins, are described in detail in Current Protocols in MolecularBiology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992,or subsequent updates thereto.

The term “host cell” is used to refer to a cell into which has beenintroduced, or which is capable of having introduced into it, a nucleicacid sequence encoding one or more of the herein described polypeptides,and which further expresses or is capable of expressing a selected geneof interest, such as a gene encoding any herein described polypeptide.The term includes the progeny of the parent cell, whether or not theprogeny are identical in morphology or in genetic make-up to theoriginal parent, so long as the selected gene is present. Accordinglythere is also contemplated a method comprising introducing such nucleicacid into a host cell. The introduction may employ any availabletechnique. For eukaryotic cells, suitable techniques may include calciumphosphate transfection, DEAE-Dextran, electroporation, liposome-mediatedtransfection and transduction using retrovirus or other virus, e.g.vaccinia or, for insect cells, baculovirus. For bacterial cells,suitable techniques may include calcium chloride transformation,electroporation and transfection using bacteriophage. The introductionmay be followed by causing or allowing expression from the nucleic acid,e.g. by culturing host cells under conditions for expression of thegene. In one embodiment, the nucleic acid is integrated into the genome(e.g. chromosome) of the host cell. Integration may be promoted byinclusion of sequences which promote recombination with the genome, inaccordance-with standard techniques.

The present invention also provides, in certain embodiments, a methodwhich comprises using a construct as stated above in an expressionsystem in order to express a particular polypeptide such as a Wnytmimetic molecule as described herein. The term “transduction” is used torefer to the transfer of genes from one bacterium to another, usually bya phage. “Transduction” also refers to the acquisition and transfer ofeukaryotic cellular sequences by retroviruses. The term “transfection”is used to refer to the uptake of foreign or exogenous DNA by a cell,and a cell has been “transfected” when the exogenous DNA has beenintroduced inside the cell membrane. A number of transfection techniquesare well known in the art and are disclosed herein. See, e.g., Graham etal., 1973, Virology 52:456; Sambrook et al., 2001, MOLECULAR CLONING, ALABORATORY MANUAL, Cold Spring Harbor Laboratories; Davis et al., 1986,BASIC METHODS IN MOLECULAR BIOLOGY, Elsevier; and Chu et al., 1981, Gene13:197. Such techniques can be used to introduce one or more exogenousDNA moieties into suitable host cells.

The term “transformation” as used herein refers to a change in a cell'sgenetic characteristics, and a cell has been transformed when it hasbeen modified to contain a new DNA. For example, a cell is transformedwhere it is genetically modified from its native state. Followingtransfection or transduction, the transforming DNA may recombine withthat of the cell by physically integrating into a chromosome of thecell, or may be maintained transiently as an episomal element withoutbeing replicated, or may replicate independently as a plasmid. A cell isconsidered to have been stably transformed when the DNA is replicatedwith the division of the cell. The term “naturally occurring” or“native” when used in connection with biological materials such asnucleic acid molecules, polypeptides, host cells, and the like, refersto materials which are found in nature and are not manipulated by ahuman. Similarly, “non-naturally occurring” or “non-native” as usedherein refers to a material that is not found in nature or that has beenstructurally modified or synthesized by a human.

The terms “polypeptide” “protein” and “peptide” and “glycoprotein” areused interchangeably and mean a polymer of amino acids not limited toany particular length. The term does not exclude modifications such asmyristylation, sulfation, glycosylation, phosphorylation and addition ordeletion of signal sequences. The terms “polypeptide” or “protein” meansone or more chains of amino acids, wherein each chain comprises aminoacids covalently linked by peptide bonds, and wherein said polypeptideor protein can comprise a plurality of chains non-covalently and/orcovalently linked together by peptide bonds, having the sequence ofnative proteins, that is, proteins produced by naturally-occurring andspecifically non-recombinant cells, or genetically-engineered orrecombinant cells, and comprise molecules having the amino acid sequenceof the native protein, or molecules having deletions from, additions to,and/or substitutions of one or more amino acids of the native sequence.The terms “polypeptide” and “protein” specifically encompass Wntsurrogate molecules, Fzd binding regions thereof, LRP5/6 binding regionsthereof, antibodies and antigen-binding fragments thereof that bind to aFzd receptor or a LRP5 or LRP6 receptor disclosed herein, or sequencesthat have deletions from, additions to, and/or substitutions of one ormore amino acid of any of these polyppetides. Thus, a “polypeptide” or a“protein” can comprise one (termed “a monomer”) or a plurality (termed“a multimer”) of amino acid chains.

The term “isolated protein,” “isolated Wnt surrogate molecule or“isolated antibody” referred to herein means that a subject protein, Wntsurrogate molecule, or antibody: (1) is free of at least some otherproteins with which it would typically be found in nature; (2) isessentially free of other proteins from the same source, e.g., from thesame species, (3) is expressed by a cell from a different species; (4)has been separated from at least about 50 percent of polynucleotides,lipids, carbohydrates, or other materials with which it is associated innature; (5) is not associated (by covalent or noncovalent interaction)with portions of a protein with which the “isolated protein” isassociated in nature; (6) is operably associated (by covalent ornoncovalent interaction) with a polypeptide with which it is notassociated in nature; or (7) does not occur in nature. Such an isolatedprotein can be encoded by genomic DNA, cDNA, mRNA or other RNA, or maybe of synthetic origin, or any combination thereof. In certainembodiments, an isolated protein may comprise naturally-occurring and/orartificial polypeptide sequences. In certain embodiments, the isolatedprotein is substantially free from proteins or polypeptides or othercontaminants that are found in its natural environment that wouldinterfere with its use (therapeutic, diagnostic, prophylactic, researchor otherwise).

Amino acid sequence modification(s) of any of the polypeptides (e.g.,Wnt surrogate molecules or Fzd binding regions or LRP5/6 binding regionsthereof) described herein are contemplated. For example, it may bedesirable to improve the binding affinity and/or other biologicalproperties of the Wnt surrogate molecule. For example, amino acidsequence variants of a Wnt surrogate molecule may be prepared byintroducing appropriate nucleotide changes into a polynucleotide thatencodes the antibody, or a chain thereof, or by peptide synthesis. Suchmodifications include, for example, deletions from, and/or insertionsinto and/or substitutions of, residues within the amino acid sequencesof the antibody. Any combination of deletion, insertion, andsubstitution may be made to arrive at the final Wnt surrogate molecule,provided that the final construct possesses the desired characteristics(e.g., high affinity binding to one or more Fzd and/or LRP5/6 receptor).The amino acid changes also may alter post-translational processes ofthe antibody, such as changing the number or position of glycosylationsites. Any of the variations and modifications described above forpolypeptides of the present invention may be included in antibodies ofthe present invention.

The present disclosure provides variants of any of the polypeptides(e.g., Wnt surrogate molecules or Fzd binding regions or LRP5/6 bindingregions thereof, or antibodies or antigen-binding fragments thereof)disclosed herein. In certain embodiments, a variant has at least 90%, atleast 95%, at least 98%, or at least 99% identity to a polypeptidedisclosed herein. In certain embodiments, such variant polypeptides bindto one or more Fzd receptor, and/or to one or more LRP5/6 receptor, atleast about 50%, at least about 70%, and in certain embodiments, atleast about 90% as well as a Wnt surrogate molecule specifically setforth herein. In further embodiments, such variant Wnt surrogatemolecules bind to one or more Fzd receptor, and/or to one or more LRP5/6receptor, with greater affinity than the Wnt surrogate molecules setforth herein, for example, that bind quantitatively at least about 105%,106%, 107%, 108%, 109%, or 110% as well as an antibody sequencespecifically set forth herein.

In particular embodiments, the Wnt surrogate molecule or a bindingregion thereof, e.g., a Fab, scFv, or VHH or sdAb may comprise: a) aheavy chain variable region comprising: i. a CDR1 region that isidentical in amino acid sequence to the heavy chain CDR1 region of aselected antibody described herein; ii. a CDR2 region that is identicalin amino acid sequence to the heavy chain CDR2 region of the selectedantibody; and iii. a CDR3 region that is identical in amino acidsequence to the heavy chain CDR3 region of the selected antibody; and/orb) a light chain variable domain comprising: i. a CDR1 region that isidentical in amino acid sequence to the light chain CDR1 region of theselected antibody; ii. a CDR2 region that is identical in amino acidsequence to the light chain CDR2 region of the selected antibody; andiii. a CDR3 region that is identical in amino acid sequence to the lightchain CDR3 region of the selected antibody; wherein the antibodyspecifically binds a selected target (e.g., one or more Fzd receptors orLRP5 or LRP6 receptors). In a further embodiment, the antibody, orantigen-binding fragment thereof, is a variant antibody orantigen-binding fragment thereof wherein the variant comprises a heavyand light chain identical to the selected antibody except for up to 8,9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions in the CDRregions of the VH and VL regions. In this regard, there may be 1, 2, 3,4, 5, 6, 7, 8, or in certain embodiments, 9, 10, 11, 12, 13, 14, 15 moreamino acid substitutions in the CDR regions of the selected antibody.Substitutions may be in CDRs either in the VH and/or the VL regions.(See e.g., Muller, 1998, Structure 6:1153-1167).

In particular embodiments, the Wnt surrogate molecule or a bindingregion thereof, e.g., a Fab, scFv, or VHH or sdAb may have: a) a heavychain variable region having an amino acid sequence that is at least 80%identical, at least 95% identical, at least 90%, at least 95% or atleast 98% or 99% identical, to the heavy chain variable region of anantibody or antigen-binding fragments thereof described herein; and/orb) a light chain variable region having an amino acid sequence that isat least 80% identical, at least 85%, at least 90%, at least 95% or atleast 98% or 99% identical, to the light chain variable region of anantibody or antigen-binding fragments thereof described herein. Theamino acid sequence of illustrative antigen-binding fragments thereofare set forth in SEQ ID NOs:1-128.

A polypeptide has a certain percent “sequence identity” to anotherpolypeptide, meaning that, when aligned, that percentage of amino acidsare the same when comparing the two sequences. Sequence similarity canbe determined in a number of different manners. To determine sequenceidentity, sequences can be aligned using the methods and computerprograms, including BLAST, available over the world wide web atncbi.nlm.nih.gov/BLAST/. Another alignment algorithm is FASTA, availablein the Genetics Computing Group (GCG) package, from Madison, Wis., USA,a wholly owned subsidiary of Oxford Molecular Group, Inc. Othertechniques for alignment are described in Methods in Enzymology, vol.266: Computer Methods for Macromolecular Sequence Analysis (1996), ed.Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., SanDiego, Calif., USA. Of particular interest are alignment programs thatpermit gaps in the sequence. The Smith-Waterman is one type of algorithmthat permits gaps in sequence alignments. See Meth. Mol. Biol. 70:173-187 (1997). Also, the GAP program using the Needleman and Wunschalignment method can be utilized to align sequences. See J. Mol. Biol.48: 443-453 (1970)

Of interest is the BestFit program using the local homology algorithm ofSmith and Waterman (Advances in Applied Mathematics 2: 482-489 (1981) todetermine sequence identity. The gap generation penalty will generallyrange from 1 to 5, usually 2 to 4 and in many embodiments will be 3. Thegap extension penalty will generally range from about 0.01 to 0.20 andin many instances will be 0.10. The program has default parametersdetermined by the sequences inputted to be compared. Preferably, thesequence identity is determined using the default parameters determinedby the program. This program is available also from Genetics ComputingGroup (GCG) package, from Madison, Wis., USA.

Another program of interest is the FastDB algorithm. FastDB is describedin Current Methods in Sequence Comparison and Analysis, MacromoleculeSequencing and Synthesis, Selected Methods and Applications, pp.127-149, 1988, Alan R. Liss, Inc. Percent sequence identity iscalculated by FastDB based upon the following parameters:

Mismatch Penalty: 1.00; Gap Penalty: 1.00; Gap Size Penalty: 0.33; andJoining Penalty: 30.0.

In particular embodiments, the Wnt surrogate molecule or a bindingregion thereof, e.g., a Fab, scFv, or VHH or sdAb may comprise: a) aheavy chain variable region comprising: i. a CDR1 region that isidentical in amino acid sequence to the heavy chain CDR1 region of aselected antibody described herein; ii. a CDR2 region that is identicalin amino acid sequence to the heavy chain CDR2 region of the selectedantibody; and iii. a CDR3 region that is identical in amino acidsequence to the heavy chain CDR3 region of the selected antibody; and b)a light chain variable domain comprising: i. a CDR1 region that isidentical in amino acid sequence to the light chain CDR1 region of theselected antibody; ii. a CDR2 region that is identical in amino acidsequence to the light chain CDR2 region of the selected antibody; andiii. a CDR3 region that is identical in amino acid sequence to the lightchain CDR3 region of the selected antibody; wherein the antibodyspecifically binds a selected target (e.g., a Fzd receptor, such asFzd1). In a further embodiment, the antibody, or antigen-bindingfragment thereof, is a variant antibody wherein the variant comprises aheavy and light chain identical to the selected antibody except for upto 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions in theCDR regions of the VH and VL regions. In this regard, there may be 1, 2,3, 4, 5, 6, 7, 8, or in certain embodiments, 9, 10, 11, 12, 13, 14, 15more amino acid substitutions in the CDR regions of the selectedantibody. Substitutions may be in CDRs either in the VH and/or the VLregions. (See e.g., Muller, 1998, Structure 6:1153-1167).

Determination of the three-dimensional structures of representativepolypeptides (e.g., variant Fzd binding regions or LRP5/6 bindingregions of Wnt surrogate molecules as provided herein) may be madethrough routine methodologies such that substitution, addition, deletionor insertion of one or more amino acids with selected natural ornon-natural amino acids can be virtually modeled for purposes ofdetermining whether a so derived structural variant retains thespace-filling properties of presently disclosed species. See, forinstance, Donate et al., 1994 Prot. Sci. 3:2378; Bradley et al., Science309: 1868-1871 (2005); Schueler-Furman et al., Science 310:638 (2005);Dietz et al., Proc. Nat. Acad. Sci. USA 103:1244 (2006); Dodson et al.,Nature 450:176 (2007); Qian et al., Nature 450:259 (2007); Raman et al.Science 327:1014-1018 (2010). Some additional non-limiting examples ofcomputer algorithms that may be used for these and related embodiments,such as for rational design of binding regions include VMD which is amolecular visualization program for displaying, animating, and analyzinglarge biomolecular systems using 3-D graphics and built-in scripting(see the website for the Theoretical and Computational Biophysics Group,University of Illinois at Urbana-Champagne, atks.uiuc.edu/Research/vmd/. Many other computer programs are known in theart and available to the skilled person and which allow for determiningatomic dimensions from space-filling models (van der Waals radii) ofenergy-minimized conformations; GRID, which seeks to determine regionsof high affinity for different chemical groups, thereby enhancingbinding, Monte Carlo searches, which calculate mathematical alignment,and CHARMM (Brooks et al. (1983) J. Comput. Chem. 4:187-217) and AMBER(Weiner et al (1981) J. Comput. Chem. 106: 765), which assess forcefield calculations, and analysis (see also, Eisenfield et al. (1991) Am.J. Physiol. 261:C376-386; Lybrand (1991) J. Pharm. Belg. 46:49-54;Froimowitz (1990) Biotechniques 8:640-644; Burbam et al. (1990) Proteins7:99-111; Pedersen (1985) Environ. Health Perspect. 61:185-190; and Kiniet al. (1991) J. Biomol. Struct. Dyn. 9:475-488). A variety ofappropriate computational computer programs are also commerciallyavailable, such as from Schrödinger (Munich, Germany).

Compositions

Pharmaceutical compositions comprising a Wnt surrogate moleculedescribed herein and one or more pharmaceutically acceptable diluent,carrier, or excipient are also disclosed. In particular embodiments, thepharmaceutical composition further comprises one or more Wntpolypeptides or Norrin polypeptides.

In further embodiments, pharmaceutical compositions comprising apolynucleotide comprising a nucleic acid sequence encoding a Wntsurrogate molecule described herein and one or more pharmaceuticallyacceptable diluent, carrier, or excipient are also disclosed. Inparticular embodiments, the pharmaceutical composition further comprisesone or more polynucleotides comprising a nucleic acid sequence encodinga Wnt polypeptide or Norrin polypeptide. In certain embodiments, thepolynucleotides are DNA or mRNA, e.g., a modified mRNA. In particularembodiments, the polynucleotides are modified mRNAs further comprising a5′ cap sequence and/or a 3′ tailing sequence, e.g., a polyA tail. Inother embodiments, the polynucleotides are expression cassettescomprising a promoter operatively linked to the coding sequences. Incertain embodiments, the nucleic acid sequence encoding the Wntsurrogate molecule and the nucleic acid sequence encoding the Wntpolypeptide or Norrin polypeptide are present in the samepolynucleotide.

In further embodiments, pharmaceutical compositions comprising anexpression vector, e.g., a viral vector, comprising a polynucleotidecomprising a nucleic acid sequence encoding a Wnt surrogate moleculedescribed herein and one or more pharmaceutically acceptable diluent,carrier, or excipient are also disclosed. In particular embodiments, thepharmaceutical composition further comprises an expression vector, e.g.,a viral vector, comprising a polynucleotide comprising a nucleic acidsequence encoding a Wnt polypeptide or Norrin polypeptide. In certainembodiments, the nucleic acid sequence encoding the Wnt surrogatemolecule and the nucleic acid sequence encoding the Wnt polypeptide orNorrin polypeptide are present in the same polynucleotide, e.g.,expression cassette.

The present invention further contemplates a pharmaceutical compositioncomprising a cell comprising an expression vector comprising apolynucleotide comprising a promoter operatively linked to a nucleicacid encoding a Wnt surrogate molecule and one or more pharmaceuticallyacceptable diluent, carrier, or excipient. In particular embodiments,the pharmaceutical composition further comprises a cell comprising anexpression vector comprising a polynucleotide comprising a promoteroperatively linked to a nucleic acid sequence encoding a Wnt polypeptideor a Norrin polypeptide. In certain embodiments, the nucleic acidsequence encoding the Wnt surrogate molecule and the nucleic acidsequence encoding the Wnt polypeptide or Norrin polypeptide are presentin the same polynucleotide, e.g., expression cassette and/or in the samecell. In particular embodiments, the cell is a heterologous cell or anautologous cell obtained from the subject to be treated. In particularembodiments, the cell is a stem cell, e.g., an adipose-derived stem cellor a hematopoietic stem cell.

The present disclosure contemplates pharmaceutical compositionscomprising a first molecule for delivery of a Wnt surrogate molecule asa first active agent and a second molecule for delivery of a Wntpolypeptide or Norrin polypeptide. The first and second molecule may bethe same type of molecule or different types of molecules. For example,in certain embodiments, the first and second molecule may each beindependently selected from the following types of molecules:polypeptides, small organic molecules, nucleic acids encoding the firstor second active agent (optionally DNA or mRNA, optionally modifiedRNA), vectors comprising a nucleic acid sequence encoding the first orsecond active agent (optionally expression vectors or viral vectors),and cells comprising a nucleic acid sequence encoding the first orsecond active agent (optionally an expression cassette).

The subject molecules, alone or in combination, can be combined withpharmaceutically-acceptable carriers, diluents, excipients and reagentsuseful in preparing a formulation that is generally safe, non-toxic, anddesirable, and includes excipients that are acceptable for mammalian,e.g., human or primate, use. Such excipients can be solid, liquid,semisolid, or, in the case of an aerosol composition, gaseous. Examplesof such carriers, diluents and excipients include, but are not limitedto, water, saline, Ringer's solutions, dextrose solution, and 5% humanserum albumin. Supplementary active compounds can also be incorporatedinto the formulations. Solutions or suspensions used for theformulations can include a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial compounds such asbenzyl alcohol or methyl parabens; antioxidants such as ascorbic acid orsodium bisulfite; chelating compounds such as ethylenediaminetetraaceticacid (EDTA); buffers such as acetates, citrates or phosphates;detergents such as Tween 20 to prevent aggregation; and compounds forthe adjustment of tonicity such as sodium chloride or dextrose. The pHcan be adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. In particular embodiments, the pharmaceutical compositionsare sterile.

Pharmaceutical compositions may further include sterile aqueoussolutions or dispersions and sterile powders for the extemporaneouspreparation of sterile injectable solutions or dispersion. Forintravenous administration, suitable carriers include physiologicalsaline, bacteriostatic water, or phosphate buffered saline (PBS). Insome cases, the composition is sterile and should be fluid such that itcan be drawn into a syringe or delivered to a subject from a syringe. Incertain embodiments, it is stable under the conditions of manufactureand storage and is preserved against the contaminating action ofmicroorganisms such as bacteria and fungi. The carrier can be, e.g., asolvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), and suitable mixtures thereof. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, sodium chloride in the composition. Prolongedabsorption of the internal compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin.

Sterile solutions can be prepared by incorporating the anti-Fzd antibodyor antigen-binding fragment thereof (or encoding polynucleotide or cellcomprising the same) in the required amount in an appropriate solventwith one or a combination of ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the active compound into a sterile vehicle thatcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, methods of preparation arevacuum drying and freeze-drying that yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

In one embodiment, the pharmaceutical compositions are prepared withcarriers that will protect the antibody or antigen-binding fragmentthereof against rapid elimination from the body, such as a controlledrelease formulation, including implants and microencapsulated deliverysystems. Biodegradable, biocompatible polymers can be used, such asethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations will be apparent to those skilled in the art. The materialscan also be obtained commercially. Liposomal suspensions can also beused as pharmaceutically acceptable carriers. These can be preparedaccording to methods known to those skilled in the art.

It may be advantageous to formulate the pharmaceutical compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of active antibody orantigen-binding fragment thereof calculated to produce the desiredtherapeutic effect in association with the required pharmaceuticalcarrier. The specification for the dosage unit forms are dictated by anddirectly dependent on the unique characteristics of the antibody orantigen-binding fragment thereof and the particular therapeutic effectto be achieved, and the limitations inherent in the art of compoundingsuch an active antibody or antigen-binding fragment thereof for thetreatment of individuals.

The pharmaceutical compositions can be included in a container, pack, ordispenser, e.g. syringe, e.g. a prefilled syringe, together withinstructions for administration.

The pharmaceutical compositions of the invention encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other compound which, upon administration to an animal comprising ahuman, is capable of providing (directly or indirectly) the biologicallyactive antibody or antigen-binding fragment thereof.

The present invention includes pharmaceutically acceptable salts of aWnt surrogate molecule described herein. The term “pharmaceuticallyacceptable salt” refers to physiologically and pharmaceuticallyacceptable salts of the compounds of the invention: i.e., salts thatretain the desired biological activity of the parent compound and do notimpart undesired toxicological effects thereto. A variety ofpharmaceutically acceptable salts are known in the art and described,e.g., in “Remington's Pharmaceutical Sciences”, 17th edition, Alfonso R.Gennaro (Ed.), Mark Publishing Company, Easton, Pa., USA, 1985 (and morerecent editions thereof), in the “Encyclopaedia of PharmaceuticalTechnology”, 3rd edition, James Swarbrick (Ed.), Informa Healthcare USA(Inc.), NY, USA, 2007, and in J. Pharm. Sci. 66: 2 (1977). Also, for areview on suitable salts, see “Handbook of Pharmaceutical Salts:Properties, Selection, and Use” by Stahl and Wermuth (Wiley-VCH, 2002).

Pharmaceutically acceptable base addition salts are formed with metalsor amines, such as alkali and alkaline earth metals or organic amines.Metals used as cations comprise sodium, potassium, magnesium, calcium,and the like. Amines comprise N—N′-dibenzylethylenediamine,chloroprocaine, choline, diethanolamine, dicyclohexylamine,ethylenediamine, N-methylglucamine, and procaine (see, for example,Berge et al., “Pharmaceutical Salts,” J. Pharma Sci., 1977, 66, 119).The base addition salts of said acidic compounds are prepared bycontacting the free acid form with a sufficient amount of the desiredbase to produce the salt in the conventional manner. The free acid formmay be regenerated by contacting the salt form with an acid andisolating the free acid in the conventional manner. The free acid formsdiffer from their respective salt forms somewhat in certain physicalproperties such as solubility in polar solvents, but otherwise the saltsare equivalent to their respective free acid for purposes of the presentinvention.

In some embodiments, the pharmaceutical composition provided hereincomprise a therapeutically effective amount of a Wnt surrogate moleculeor pharmaceutically acceptable salt thereof in admixture with apharmaceutically acceptable carrier, diluent and/or excipient, forexample saline, phosphate buffered saline, phosphate and amino acids,polymers, polyols, sugar, buffers, preservatives and other proteins.Exemplary amino acids, polymers and sugars and the like are octylphenoxypolyethoxy ethanol compounds, polyethylene glycol monostearatecompounds, polyoxyethylene sorbitan fatty acid esters, sucrose,fructose, dextrose, maltose, glucose, mannitol, dextran, sorbitol,inositol, galactitol, xylitol, lactose, trehalose, bovine or human serumalbumin, citrate, acetate, Ringer's and Hank's solutions, cysteine,arginine, camitine, alanine, glycine, lysine, valine, leucine,polyvinylpyrrolidone, polyethylene and glycol. Preferably, thisformulation is stable for at least six months at 4° C.

In some embodiments, the pharmaceutical composition provided hereincomprises a buffer, such as phosphate buffered saline (PBS) or sodiumphosphate/sodium sulfate, tris buffer, glycine buffer, sterile water andother buffers known to the ordinarily skilled artisan such as thosedescribed by Good et al. (1966) Biochemistry 5:467. The pH of the buffermay be in the range of 6.5 to 7.75, preferably 7 to 7.5, and mostpreferably 7.2 to 7.4.

Methods of Use

The present disclosure also provides methods for using the Wnt surrogatemolecules disclosed herein, e.g., to modulate a Wnt signaling pathway,e.g., to increase Wnt signaling, and the administration of a Wntsurrogate molecule disclosed herein in a variety of therapeuticsettings. Provided herein are methods of treatment using a Wnt surrogatemolecule. In one embodiment, a Wnt surrogate molecule is provided to asubject having a disease involving inappropriate or deregulated Wntsignaling, e.g., reduced Wnt signaling.

Increasing Wnt Pathway Signaling and Related Therapeutic Methods

In certain embodiments, a Wnt surrogate molecule may be used to increaseWnt signaling in a tissue or cell. Thus, in some aspects, the presentinvention provides a method for increasing Wnt signaling or enhancingWnt signaling in a tissue or cell, comprising contacting the tissue orcell with an effective amount of a Wnt surrogate molecule orpharmaceutically acceptable salt thereof disclosed herein, wherein the aWnt surrogate molecule is a Wnt signaling pathway agonist. In someembodiments, contacting occurs in vitro, ex vivo, or in vivo. Inparticular embodiments, the cell is a cultured cell, and the contactingoccurs in vitro. In certain embodiments, the method comprises furthercontacting the tissue or cell with one or more Wnt polypeptides orNorrin polypeptides.

In related aspects, the present invention provides a method forincreasing Wnt signaling in a tissue or cell, comprising contacting thetissue or cell with an effective amount of a polynucleotide comprising aWnt surrogate molecule disclosed herein. In certain embodiments, thetarget tissue or cell is also contacted with a polynucleotide comprisinga nucleic acid sequence that encodes a Wnt polypeptide or a Norrinpolypeptide. In certain embodiments, the polynucleotides are DNA ormRNA, e.g., a modified mRNA. In particular embodiments, thepolynucleotides are modified mRNAs further comprising a 5′ cap sequenceand/or a 3′ tailing sequence, e.g., a polyA tail. In other embodiments,the polynucleotides are expression cassettes comprising a promoteroperatively linked to the coding sequences. In certain embodiments, thenucleic acid sequence encoding the Wnt surrogate molecule and thenucleic acid sequence encoding the Wnt polypeptide or Norrin polypeptideare present in the same polynucleotide.

In related aspects, the present invention provides a method forincreasing Wnt signaling in a tissue or cell, comprising contacting thetissue or cell with an effective amount of a vector comprising a nucleicacid sequence encoding a Wnt surrogate molecule. In certain embodiments,the tissue or cell is also contacted with a vector comprising a nucleicacid sequence that encodes a Wnt polypeptide or a Norrin polypeptide. Incertain embodiments, the vector is an expression vector, and maycomprise a promoter operatively linked to the nucleic acid sequence. Inparticular embodiments, the vector is a viral vector. In certainembodiments, the nucleic acid sequence encoding a Wnt surrogate moleculeand the nucleic acid sequence encoding the Wnt polypeptide or Norrinpolypeptide are present in the same vector, e.g., in the same expressioncassette.

In related aspects, the present invention provides a method forincreasing Wnt signaling in a tissue, comprising contacting the tissuewith an effective amount of a cell comprising a nucleic acid sequenceencoding a Wnt surrogate molecule of the present invention. In certainembodiments, the tissue is also contacted with a cell comprising anucleic acid sequence that encodes a Wnt polypeptide or Norrinpolypeptide. In certain embodiments, the nucleic acid sequence encodingthe Wnt surrogate molecule and the nucleic acid sequence encoding theWnt polypeptide or Norrin polypeptide are present in the same cell. Inparticular embodiments, the cell is a heterologous cell or an autologouscell obtained from the subject to be treated. In certain embodiments,the cell was transduced with a vector comprising an expression cassetteencoding the Wnt surrogate molecule or the Wnt polypeptide or Norrinpolypeptide. In particular embodiments, the cell is a stem cell, e.g.,an adipose-derived stem cell or a hematopoietic stem cell.

Wnt surrogate molecules disclosed herein may be used in to treat adisease, disorder or condition, for example, by increasing Wnt signalingin a targeted cell, tissue or organ. Thus, in some aspects, the presentinvention provides a method for treating a disease or condition in asubject in need thereof, e.g., a disease or disorder associated withreduced Wnt signaling, or for which increased Wnt signaling wouldprovide a therapeutic benefit, comprising contacting the subject with aneffective amount of a composition of the present disclosure. Inparticular embodiments, the composition is a pharmaceutical compositioncomprising any of: a Wnt surrogate molecule; a polynucleotide comprisinga nucleic acid sequence encoding a Wnt surrogate molecule, e.g., a DNAor mRNA, optionally a modified mRNA; a vector comprising a nucleic acidsequence encoding a Wnt surrogate molecule, e.g., an expression vectoror viral vector; or a cell comprising a nucleic acid sequence encoding aWnt surrogate molecule, e.g., a cell transduced with an expressionvector or viral vector encoding a Wnt surrogate molecule. In particularembodiments, the disease or condition is a pathological disease ordisorder, or an injury, e.g., an injury resulting from a wound. Incertain embodiments, the wound may be the result of another therapeutictreatment. In certain embodiments, the disease or condition comprisesimpaired tissue repair, healing or regeneration, or would benefit fromincreased tissue repair, healing or regeneration. In some embodiments,contacting occurs in vivo, i.e., the subject composition is administeredto a subject.

In certain embodiments, the method comprises further contacting thesubject with a pharmaceutical composition comprising one or more Wntpolypeptides or Norrin polypeptides. The present disclosure contemplatescontacting a subject with a first molecule for delivery of a Wntsurrogate molecule as a first active agent and a second molecule fordelivery of a Wnt polypeptide or Norrin polypeptide. The first andsecond molecule may be the same type of molecule or different types ofmolecules. For example, in certain embodiments, the first and secondmolecule may each be independently selected from the following types ofmolecules: polypeptides, small organic molecules, nucleic acids encodingthe first or second active agent (optionally DNA or mRNA, optionallymodified RNA), vectors comprising a nucleic acid sequence encoding thefirst or second active agent (optionally expression vectors or viralvectors), and cells comprising a nucleic acid sequence encoding thefirst or second active agent (optionally an expression cassette).

In related aspects, the present invention provides a method for treatinga disease or condition, e.g., a disease or disorder associated withreduced Wnt signaling, or for which increased Wnt signaling wouldprovide a therapeutic benefit, comprising contacting a subject in needthereof with a pharmaceutical composition comprising an effective amountof a polynucleotide comprising a nucleic acid sequence encoding a Wntsurrogate molecule disclosed herein. In certain embodiments, the subjectis also contacted with a pharmaceutical composition comprising aneffective amount of a polynucleotide comprising a nucleic acid sequencethat encodes a Wnt polypeptide or a Norrin polypeptide. In certainembodiments, the polynucleotides are DNA or mRNA, e.g., a modified mRNA.In particular embodiments, the polynucleotides are modified mRNAsfurther comprising a 5′ cap sequence and/or a 3′ tailing sequence, e.g.,a polyA tail. In other embodiments, the polynucleotides are expressioncassettes comprising a promoter operatively linked to the codingsequences. In certain embodiments, the nucleic acid sequence encodingthe Wnt surrogate molecule and the nucleic acid sequence encoding theWnt polypeptide or Norrin polypeptide are present in the samepolynucleotide.

In related aspects, the present invention provides a method for treatinga disease or condition, e.g., a disease or disorder associated withreduced Wnt signaling, or for which increased Wnt signaling wouldprovide a therapeutic benefit, comprising contacting a subject in needthereof with a pharmaceutical composition comprising an effective amountof a vector comprising a nucleic acid sequence encoding a Wnt surrogatemolecule. In certain embodiments, the subject is also contacted with apharmaceutical composition comprising an effective amount of a vectorcomprising a nucleic acid sequence that encodes a Wnt polypeptide or aNorrin polypeptide. In certain embodiments, the vector is an expressionvector, and may comprise a promoter operatively linked to the nucleicacid sequence. In particular embodiments, the vector is a viral vector.In certain embodiments, the nucleic acid sequence encoding the Wntsurrogate molecule and the nucleic acid sequence encoding the Wntpolypeptide or Norrin polypeptide are present in the same vector, e.g.,in the same expression cassette.

In related aspects, the present invention provides a method for treatinga disease or condition, e.g., a disease or disorder associated withreduced Wnt signaling, or for which increased Wnt signaling wouldprovide a therapeutic benefit, comprising contacting a subject in needthereof with a pharmaceutical composition comprising an effective amountof a cell comprising a nucleic acid sequence encoding a Wnt surrogatemolecule. In certain embodiments, the subject is also contacted with acell comprising a nucleic acid sequence that encodes a Wnt polypeptideor a Norrin polypeptide. In certain embodiments, the nucleic acidsequence encoding the Wnt surrogate molecule and the nucleic acidsequence encoding the Wnt polypeptide or Norrin polypeptide are presentin the same cell. In particular embodiments, the cell is a heterologouscell or an autologous cell obtained from the subject to be treated. Incertain embodiments, the cell was transduced with a vector comprising anexpression cassette encoding the Wnt surrogate molecule or the Wntpolypeptide or Norrin polypeptide. In particular embodiments, the cellis a stem cell, e.g., an adipose-derived stem cell or a hematopoieticstem cell.

Wnt signaling plays key roles in the developmental process andmaintenance of stem cells. Reactivation of Wnt signals is associatedwith regeneration and repair of most tissues after injuries anddiseases. Wnt surrogate molecule molecules are expected to providebenefit of healing and tissue repair in response to injuries anddiseases. Causes of tissue damage and loss include but are not limitedto aging, degeneration, hereditary conditions, infection andinflammation, traumatic injuries, toxins/metabolic-induced toxicities,or other pathological conditions. Wnt signals and enhancers of Wntsignals have been shown to activate adult, tissue-resident stem cells.In some embodiments, the compounds of the invention are administered foruse in treating diseased or damaged tissue, for use in tissueregeneration and for use in cell growth and proliferation, and/or foruse in tissue engineering.

Human diseases associated with mutations of the Wnt pathway providestrong evidence for enhancement of Wnt signals in the treatment andprevention of diseases. Preclinical in vivo and in vitro studies provideadditional evidence of involvement of Wnt signals in many diseaseconditions and further support utilization of a Wnt surrogate moleculein various human diseases. For example, compositions of the presentinvention may be used to promote or increase bone growth orregeneration, bone grafting, healing of bone fractures, treatment ofosteoporosis and osteoporotic fractures, spinal fusion, spinal cordinjuries, including vertebral compression fractures, pre-operativespinal surgery optimization, osseointegration of orthopedic devices,tendon-bone integration, tooth growth and regeneration, dentalimplantation, periodontal diseases, maxillofacial reconstruction, andosteonecrosis of the jaw. They may also be used in the treatment ofalopecia; enhancing regeneration of sensory organs, e.g. treatment ofhearing loss, including regeneration of inner and outer auditory haircells treatment of vestibular hypofunction, treatment of maculardegeneration, treatment of retinopathies, including vitreoretinopathy,diabetic retinopathy, other diseases of retinal degeneration, Fuchs'dystrophy, other cornea disease, etc.; treatment of stroke, traumaticbrain injury, Alzheimer's disease, multiple sclerosis, multipledystrophy, muscle atrophy as a result of sarcopenia or cachexia, andother conditions affecting the degeneration or integrity of the bloodbrain barrier; The compositions of this invention may also be used intreatment of oral mucositis, treatment of short bowel syndrome,inflammatory bowel diseases (IBD), including Crohn's disease (CD) andulcerative colitis (UC), in particular CD with fistula formation, othergastrointestinal disorders; treatment of metabolic syndrome,dyslipidemia, treatment of diabetes, treatment of pancreatitis,conditions where exocrine or endocrine pancreas tissues are damaged;conditions where enhanced epidermal regeneration is desired, e.g.,epidermal wound healing, treatment of diabetic foot ulcers, syndromesinvolving tooth, nail, or dermal hypoplasia, etc., conditions whereangiogenesis is beneficial; treatment of myocardial infarction, coronaryartery disease, heart failure; enhanced growth of hematopoietic cells,e.g. enhancement of hematopoietic stem cell transplants from bonemarrow, mobilized peripheral blood, treatment of immunodeficiencies,graft versus host diseases, etc.; treatment of acute kidney injuries,chronic kidney diseases; treatment of lung diseases, chronic obstructivepulmonary diseases (COPD), pulmonary fibrosis, including idiopathicpulmonary fibrosis, enhanced regeneration of lung tissues. Thecompositions of the present invention may also be used in enhancedregeneration of liver cells, e.g. liver regeneration, treatment ofcirrhosis, enhancement of liver transplantations, treatment of acuteliver failure, treatment of chronic liver diseases with hepatitis C or Bvirus infection or post-antiviral drug therapies, alcoholic liverdiseases, including alcoholic hepatitis, non-alcoholic liver diseaseswith steatosis or steatohepatitis, and the like. The compositions ofthis invention may treat diseases and disorders including, withoutlimitation, conditions in which regenerative cell growth is desired.

Human genetics involving loss-of-function or gain-of-function mutationsin Wnt signaling components show strong evidence supporting enhancingWnt signals for bone growth. Conditions in which enhanced bone growth isdesired may include, without limitation, fractures, grafts, ingrowtharound prosthetic devices, osteoporosis, osteoporotic fractures, spinalfusion, vertebral compression fractures, pre-operative optimization forspinal surgeries, osteonecrosis of the jaw, dental implantation,periodontal diseases, maxillofacial reconstruction, and the like. Wntsurrogate molecules enhance and promotes Wnt signals which are criticalin promoting bone regeneration. Methods for regeneration of bone tissuesbenefit from administration of the compounds of the invention, which canbe systemic or localized. In some embodiments, bone marrow cells areexposed to molecules of the invention, such that stem cells within thatmarrow become activated.

In some embodiments, bone regeneration is enhanced by contacting aresponsive cell population, e.g. bone marrow, bone progenitor cells,bone stem cells, etc. with an effective dose of a Wnt surrogate moleculedisclosed herein. Methods for regeneration of bone tissues benefit fromadministration of the Wnt surrogate molecule which can be systemic orlocalized. In some such embodiments, the contacting is performed invivo. In other such embodiments, the contacting is performed ex vivo.The molecule may be localized to the site of action, e.g. by loadingonto a matrix, which is optionally biodegradable, and optionallyprovides for a sustained release of the active agent. Matrix carriersinclude, without limitation, absorbable collagen sponges, ceramics,hydrogels, polymeric microspheres, nanoparticles, bone cements, and thelike.

In particular embodiments, compositions comprising one or more Wntsurrogate molecule disclosed herein (or a polynucleotide encoding a Wntsurrogate molecule, or a vector or cell comprising a polynucleotideencoding a Wnt surrogate molecule) are used to treat or prevent a bonedisease or disorder, including but not limited to any of the following,or to treat or prevent an injury associated with, but not limited to,any of the following: osteoporosis, osteoporotic fractures, bonefractures including vertebral compression fractures, non-unionfractures, delayed union fractures, spinal fusion, osteonecrosis,osteonecrosis of the jaw, hip, femoral head, etc., osseointegration ofimplants (e.g., to accelerate recovery following partial or total kneeor hip replacement), osteogenesis imperfecta, bone grafts, tendonrepair, maxillofacial surgery, dental implant, all other bone disordersor defects resulting from genetic diseases, degeneration, aging, drugs,or injuries. In one embodiment, Wnt surrogate molecules that bind Fzd1,Fzd 2, and Fzd 7, and also LRP5 and/or LRP6, are used to treat orprevent any bone disease or disorder. In one embodiment, Wnt surrogatemolecules that bind Fzd1, Fzd 2, Fzd 5, Fzd 7 and Fzd 8, and also LRP5and/or LRP6, are used to treat or prevent any bone disease or disorder.Other Fzd molecules that bind to additional Fzd receptors can also beused with LRP5 and/or LRP6 binders.

In particular embodiments, compositions and methods disclosed herein maybe used to: increase bone mineral density, increase bone volume (e.g.,tibia and/or femur bone volume), increase cortical thickness (e.g., intrabecular region or in femur mid-diaphysis), increase mineralapposition rate, increase the number of osteblasts and/or decrease thenumber of osteoclasts (e.g., in bone), increase bone stiffness, increasethe ultimate load to fracture point, improve bone resistance tofracture, decrease bone resorption, decrease bone loss associated withosteoporosis, or increase biochemical strength of bone, in a subject. Inone embodiment, Wnt surrogate molecules that bind Fzd1, Fzd 2, and Fzd 7are used for any of these indicated uses. In one embodiment, Wntsurrogate molecules that bind Fzd1, Fzd 2, Fzd 5, Fzd 7 and Fzd 8 areused for any of these indicated uses.

Methods disclosed herein, including methods for treating or preventing abone disease or disorder include methods that comprise providing to asubject in need thereof both a Wnt surrogate molecule and anantiresorptive agent. In certain embodiments, the methods are used forthe treatment of osteoporosis, optionally post-menopausal osteoporosis.

The disclosure also provides a method for inhibiting or reducing boneresorption in a subject in need thereof, comprising providing to thesubject an effective amount of a Wnt surrogate molecule, wherein the Wntsurrogate molecule is an agonist of a Wnt signaling pathway. In certainembodiments, the method further comprises providing to the subject anantiresorptive agent. In certain embodiments, the subject has beendiagnosed with or is at risk for osteoporosis, optionally postmenopausalosteoporosis. A variety of antiresorptive agents are known in the artand include, but are not limited to, those disclosed herein.

When a Wnt surrogate molecule is provide to the subject in combinationwith another therapeutic agent, such as an antiresorptive agent, the twoagent may be provided in the same or different pharmaceuticalcompositions. They may be provided to the subject at the same time, atdifferent times, e.g., simultaneously, consecutively, or duringoverlapping or non-overlapping time periods. In certain embodiments, thetwo agents are therapeutically active in the subject during anoverlapping time period.

Compositions comprising one or more Wnt surrogate molecule disclosedherein (or a polynucleotide encoding a Wnt surrogate molecule, or avector or cell comprising a polynucleotide encoding a Wnt surrogatemolecule) can be used for the in vivo treatment of skeletal tissuedeficiencies. By “skeletal tissue deficiency”, it is meant a deficiencyin bone or other skeletal connective tissue at any site where it isdesired to restore the bone or connective tissue, no matter how thedeficiency originated, e.g. whether as a result of surgicalintervention, removal of tumor, ulceration, implant, fracture, or othertraumatic or degenerative conditions. The compositions of the presentinvention can be used as part of a regimen for restoring cartilagefunction to a connective tissue, for the repair of defects or lesions incartilage tissue such as degenerative wear and arthritis, trauma to thetissue, displacement of torn meniscus, meniscectomy, a luxation of ajoint by a torn ligament, malalignment of joints, bone fracture, or byhereditary disease.

A Wnt surrogate molecule may also be used for treatment of periodontaldiseases. Periodontal diseases are a leading cause of tooth loss and arelinked to multiple systemic conditions. In some embodiments, tooth orunderlying bone regeneration is enhanced by contacting a responsive cellpopulation. In some such embodiments, the contacting is performed invivo. In other such embodiments, the contacting is performed ex vivo,with subsequent implantation of the activated stem or progenitor cells.The molecule may be localized to the site of action, e.g. by loadingonto a matrix, which is optionally biodegradable, and optionallyprovides for a sustained release of the active agent. Matrix carriersinclude, without limitation, absorbable collagen sponges, ceramics,hydrogels, bone cements, polymeric microspheres, nanoparticles, and thelike.

Studies have shown that biology of Wnt signaling and R-spondins arecapable of promoting sensory hair cell regeneration in the inner earfollowing injuries, aging, or degeneration. Loss of sensory hair cellsin the inner ear involved in hearing loss or vestibular hypofunction mayalso benefit from the compositions of the invention. In the inner ear,the auditory organ houses mechanosensitive hair cells required fortranslating sound vibration to electric impulses. The vestibular organs,comprised of the semicircular canals (SSCs), the utricle, and thesaccule, also contain sensory hair cells in order to detect headposition and motion. Compositions of the present invention can be used,for example, in an infusion; in a matrix or other depot system; or othertopical application to the ear for enhancement of auditory regeneration.

A Wnt surrogate molecule may also be used in regeneration of retinaltissue. In the adult mammalian retina, Muller glia cells are capable ofregenerating retinal cells, including photoreceptors, for example afterneurotoxic injury in vivo. Wnt signaling and enhancers of Wnt signalscan promote proliferation of Muller glia-derived retinal progenitorsafter damage or during degeneration. The compositions of the inventionmay also be used in the regeneration of tissues and other cell types inthe eye. For examples age-related macular degeneration (AMD), otherretina degenerative diseases, cornea diseases, Fuchs' dystrophy,vitreoretinopathy, hereditary diseases, etc. can benefit from thecompositions of the present inventions. AMD is characterized byprogressively decreased central vision and visual acuity. Fuchs'dystrophy is characterized by progressive loss of cornea endothelialcells. Wnt signal and enhancing of Wnt signal can promote regenerationof cornea endothelium, retina epithelium, etc. in the eye tissue. Inother embodiments, compositions of the present invention can be used,for example, in an infusion; in a matrix or other depot system; or othertopical application to the eye for retinal regeneration and treatment ofmacular degeneration.

Specific populations of proliferating cells for homeostatic renewal ofhepatocytes have been identified through lineage tracing studies, forexample Axin2-positive cells in peri-central region. Lineage tracingstudies also identified additional potential liver progenitor cells,including but not limited to Lgr-positive cells. The self-renewing livercells and other populations of potential progenitor cells, includingLgr5-positive and Axin2-positive cells, are identified to be capable ofregeneration responding to Wnt signals and/or R-spondins followinginjuries. Numerous preclinical models of acute liver injury and failureand chronic liver diseases showed recovery and regeneration ofhepatocytes benefit from enhancing Wnt signals.

In certain embodiments, compositions comprising a Wnt surrogate moleculedisclosed herein (or a polynucleotide encoding a Wnt surrogate molecule,or a vector or cell comprising a polynucleotide encoding a Wnt surrogatemolecule) are used to promote liver regeneration, reduce fibrosis,and/or improve liver function. In certain embodiments, compositions andmethods disclosed herein are used to: increase liver weight, increasethe liver to body weight ratio, increase the number of PCNA and pH3positive nuclei in liver, increase expression of Ki67 and/or Cyclin D1in liver, increase liver cell proliferation and/or mitosis, decreasefibrosis following chronic liver injury, or increase hepatocytefunction.

In particular embodiments, the compositions of this invention may beused in treatment of acute liver failure, acute alcoholic liverinjuries, treatment of chronic liver diseases with hepatitis C or Bvirus infection or post-antiviral drug therapies, chronic alcoholicliver diseases, alcoholic hepatitis, non-alcoholic fatty liver diseasesand non-alcoholic steatohepatitis (NASH), treatment of cirrhosis andsevere chronic liver diseases of all causes, and enhanced regenerationof liver cells. Methods for regeneration of liver tissue benefit fromadministration of the compounds of the invention, which can be systemicor localized. These include, but are not limited to, methods of systemicadministration and methods of localized administration e.g. by injectioninto the liver tissue, by injection into veins or blood vessels leadinginto the liver, by implantation of a sustained release formulation, andthe like.

In particular embodiments, compositions comprising a Wnt surrogatemolecule disclosed herein (or a polynucleotide encoding a Wnt surrogatemolecule, or a vector or cell comprising a polynucleotide encoding a Wntsurrogate molecule) are used to treat or prevent a liver disease ordisorder, including but not limited to, or to treat or prevent a liverinjury or disorder resulting from any of the following: acute liverfailure (all causes), chronic liver failure (all causes), cirrhosis,liver fibrosis (all causes), portal hypertension, alcoholic liverdiseases including alcoholic hepatitis, nonalcoholic steatohepatisis(NASH), nonalcoholic fatty liver disease (NAFLD) (fatty liver),alcoholic hepatitis, hepatitis C virus-induced liver diseases (HCV),hepatitis B virus-induced liver diseases (HBV), other viral hepatitis(e.g., hepatitis A virus-induced liver diseases (HAV) and hepatitis Dvirus-induced liver diseases (HDV)), primary biliary cirrhosis,autoimmune hepatitis, livery surgery, liver injury, livertransplantation, “small for size” syndrome in liver surgery andtransplantation, congenital liver disease and disorders, any other liverdisorder or detect resulting from genetic diseases, degeneration, aging,drugs, or injuries.

Wnt signals play an important role in regeneration of various epithelialtissues. Various epidermal conditions benefit from treatment with thecompounds of the present invention. Mucositis occurs when there is abreakdown of the rapidly divided epithelial cells lining thegastro-intestinal tract, leaving the mucosal tissue open to ulcerationand infection. The part of the epithelial lining that covers the mouth,called the oral mucosa, is one of the most sensitive parts of the bodyand is particularly vulnerable to chemotherapy and radiation. Oralmucositis is probably the most common, debilitating complication ofcancer treatments, particularly chemotherapy and radiation. In addition,the compositions of the invention may also benefit treatment of shortbowel syndrome, inflammatory bowel diseases (IBD), or othergastrointestinal disorders. Other epidermal conditions include epidermalwound healing, diabetic foot ulcers, syndromes involving tooth, nail, ordermal hypoplasia, and the like. Molecules of the present invention maybe used in all these conditions, where regenerative cells are contactedwith compounds of the invention. Methods for regeneration of epithelialtissues benefit from administration of the compounds of the invention,which can be systemic or localized. Contacting can be, for example,topical, including intradermal, subdermal, in a gel, lotion, cream etc.applied at targeted site, etc.

In addition to skin and gastrointestinal tract, Wnt signals andenhancement and promotion of Wnt signals also play an important role inrepair and regeneration of tissues including pancreas, kidney, and lungin preclinical models. A Wnt surrogate molecule may benefit variousdisease conditions involving exocrine and endocrine pancreas, kidney, orlung. The Wnt surrogate molecules may be used in treatment of metabolicsyndrome; treatment of diabetes, treatment of acute or chronicpancreatitis, exocrine pancreatic insufficiency, treatment of acutekidney injuries, chronic kidney diseases, treatment of lung diseases,including but not limited to chronic obstructive pulmonary diseases(COPD), pulmonary fibrosis, in particular idiopathic pulmonary fibrosis(IPF), and other conditions that cause loss of lung epithelial tissues.Methods for regeneration of these tissues benefit from administration ofthe compounds of the invention, which can be systemic or localized.

Epidermal Wnt signaling, in coordination with signaling via otherdevelopment factors, is critical for adult hair follicle regeneration.Hair loss is a common problem, and androgenetic alopecia, often calledmale pattern baldness, is the most common form of hair loss in men. Insome embodiments, hair follicle regeneration is enhanced by contacting aresponsive cell population with a molecule of the present invention. Insome such embodiments, the contacting is performed in vivo. In othersuch embodiments, the contacting is performed ex vivo. The molecule maybe localized to the site of action, e.g. topical lotions, gels, creamsand the like.

Stroke, traumatic brain injury, Alzheimer's disease, multiple sclerosisand other conditions affecting the blood brain barrier (BBB) may betreated with a Wnt surrogate molecule. Angiogenesis is critical toensure the supply of oxygen and nutrients to many tissues throughout thebody, and is especially important for the CNS as the neural tissue isextremely sensitive to hypoxia and ischemia. CNS endothelial cells whichform the BBB differ from endothelial cells in non-neural tissue, in thatthey are highly polarized cells held together by tight junctions andexpress specific transporters. Wnt signaling regulates CNS vesselformation and/or function. Conditions in which the BBB is compromisedcan benefit from administration of the compounds of the invention, whichcan be systemic or localized e.g. by direct injection, intrathecaladministration, implantation of sustained release formulations, and thelike. In addition, Wnt signal is actively involved in neurogenesis andplays a role of neuroprotection following injury. The compositions ofthe present invention may also be used in treatment of spinal cordinjuries, other spinal cord diseases, stroke, traumatic brain injuries,etc.

Wnt signals also play a role in angiogenesis. A Wnt surrogate moleculemay benefit conditions where angiogenesis is beneficial, treatment ofmyocardial infarction, coronary artery disease, heart failure, diabeticretinopathy, etc., and conditions from hereditary diseases. Methods forregeneration of these tissues benefit from administration of thecompounds of the invention, which can be systemic or localized.

In certain embodiments, methods of the present invention promote tissueregeneration, e.g., in a tissue subjected to damage or tissue or cellreduction or loss. The loss or damage can be anything which causes thecell number to diminish, including diseases or injuries. For example, anaccident, an autoimmune disorder, a therapeutic side-effect or a diseasestate could constitute trauma. Tissue regeneration increases the cellnumber within the tissue and preferably enables connections betweencells of the tissue to be re-established, and more preferably thefunctionality of the tissue to be regained.

The terms “administering” or “introducing” or “providing”, as usedherein, refer to delivery of a composition to a cell, to cells, tissuesand/or organs of a subject, or to a subject. Such administering orintroducing may take place in vivo, in vitro or ex vivo.

In particular embodiments, a pharmaceutical composition is administeredparenterally, e.g., intravenously, orally, rectally, or by injection. Insome embodiments, it is administered locally, e.g., topically orintramuscularly. In some embodiments, a composition is administered totarget tissues, e.g., to bone, joints, ear tissue, eye tissue,gastrointestinal tract, skin, a wound site or spinal cord. Methods ofthe invention may be practiced in vivo or ex vivo. In some embodiments,the contacting of a target cell or tissue with a Wnt surrogate moleculeis performed ex vivo, with subsequent implantation of the cells ortissues, e.g., activated stem or progenitor cells, into the subject. Theskilled artisan can determine an appropriate site of and route ofadministration based on the disease or disorder being treated.

The dose and dosage regimen may depend upon a variety of factors readilydetermined by a physician, such as the nature of the disease ordisorder, the characteristics of the subject, and the subject's history.In particular embodiments, the amount of a Wnt surrogate moleculeadministered or provided to the subject is in the range of about 0.01mg/kg to about 50 mg/kg, 0.1 mg/kg to about 500 mg/kg, or about 0.1mg/kg to about 50 mg/kg of the subject's body weight.

The terms “treatment”, “treating” and the like are used herein togenerally mean obtaining a desired pharmacologic and/or physiologiceffect. The effect may be prophylactic in terms of completely orpartially preventing a disease or symptom thereof, e.g. reducing thelikelihood that the disease or symptom thereof occurs in the subject,and/or may be therapeutic in terms of a partial or complete cure for adisease and/or adverse effect attributable to the disease. “Treatment”as used herein covers any treatment of a disease in a mammal, andincludes: (a) preventing the disease from occurring in a subject whichmay be predisposed to the disease but has not yet been diagnosed ashaving it; (b) inhibiting the disease, i.e., arresting its development;or (c) relieving the disease, i.e., causing regression of the disease.The therapeutic agent (e.g., a Wnt surrogate molecule) may beadministered before, during or after the onset of disease or injury. Thetreatment of ongoing disease, where the treatment stabilizes or reducesthe undesirable clinical symptoms of the patient, is of particularinterest. Such treatment is desirably performed prior to complete lossof function in the affected tissues. The subject therapy will desirablybe administered during the symptomatic stage of the disease, and in somecases after the symptomatic stage of the disease. In some embodiments,the subject method results in a therapeutic benefit, e.g., preventingthe development of a disorder, halting the progression of a disorder,reversing the progression of a disorder, etc. In some embodiments, thesubject method comprises the step of detecting that a therapeuticbenefit has been achieved. The ordinarily skilled artisan willappreciate that such measures of therapeutic efficacy will be applicableto the particular disease being modified, and will recognize theappropriate detection methods to use to measure therapeutic efficacy.

Other embodiments relate, in part, to the use of the Wnt surrogatemolecules disclosed herein to promote or enhance the growth orproliferation of cells, tissues and organoids, for example, bycontacting cells or tissue with one or more Wnt surrogate, optionally incombination with a Norrin or Rspondin polypeptide. In certainembodiments, the cells or tissue are contacted ex vivo, in vitro, or invivo. Such methods may be used to generate cells, tissue or organoidsfor therapeutic use, e.g., to be transplanted or grafted into a subject.They may also be used to generate cells, tissue or organoids forresearch use. The Wnt surrogate molecules have widespread applicationsin non-therapeutic methods, for example in vitro research methods.

The invention provides a method for tissue regeneration of damagedtissue, such as the tissues discussed above, comprising administering aWnt surrogate molecule to cells. The Wnt surrogate molecule may beadministered directly to the cells in vivo, administered to a subjectorally, intravenously, or by other methods known in the art, oradministered to ex vivo cells. In some embodiments where the Wntsurrogate molecule is administered to ex vivo cells, these cells may betransplanted into a subject before, after or during administration ofthe Wnt surrogate molecule.

Wnt signaling is a key component of stem cell culture. For example, thestem cell culture media as described in WO2010/090513, WO2012/014076,Sato et al., 2011 (GASTROENTEROLOGY 2011; 141: 1762-1772) and Sato etal., 2009 (Nature 459, 262-5). The Wnt surrogate molecules disclosedherein are suitable alternatives to Rspondin for use in these stem cellculture media, or may be combined with Rspondin.

Accordingly, in one embodiment, the disclosure provides a method forenhancing the proliferation of stem cells comprising contacting stemcells with one or more Wnt surrogate molecules disclosed herein. In oneembodiment, the disclosure provides a cell culture medium comprising oneor more Wnt surrogate molecules disclosed herein. In some embodiments,the cell culture medium may be any cell culture medium already known inthe art that normally comprises Wnt or Rspondin, but wherein the Wnt orRspondin is replaced (wholly or partially) or supplemented by Wntsurrogate molecule(s) disclosed herein. For example, the culture mediummay be as described in as described in WO2010/090513, WO2012/014076,Sato et al., 2011 (GASTROENTEROLOGY 2011; 141: 1762-1772) and Sato etal., 2009 (Nature 459, 262-5), which are hereby incorporated byreference in their entirety.

Stem cell culture media often comprise additional growth factors. Thismethod may thus additionally comprise supplying the stem cells with agrowth factor. Growth factors commonly used in cell culture mediuminclude epidermal growth factor (EGF, (Peprotech), Transforming GrowthFactor-alpha (TGF-alpha, Peprotech), basic Fibroblast Growth Factor(bFGF, Peprotech), brain-derived neurotrophic factor (BDNF, R&DSystems), Hepatocyte Growth Factor (HGF) and Keratinocyte Growth Factor(KGF, Peprotech, also known as FGF7). EGF is a potent mitogenic factorfor a variety of cultured ectodermal and mesodermal cells and has aprofound effect on the differentiation of specific cells in vivo and invitro and of some fibroblasts in cell culture. The EGF precursor existsas a membrane-bound molecule which is proteolytically cleaved togenerate the 53-amino acid peptide hormone that stimulates cells. EGF orother mitogenic growth factors may thus be supplied to the stem cells.During culturing of stem cells, the mitogenic growth factor may be addedto the culture medium every second day, while the culture medium isrefreshed preferably every fourth day. In general, a mitogenic factor isselected from the groups consisting of: i) EGF, TGF-alpha, and KGF, ii)EGF, TGF-alpha, and FGF7; iii) EGF, TGF-alpha, and FGF; iv) EGF and KGF;v) EGF and FGF7; vi) EGF and a FGF; vii) TGF-alpha and KGF; viii)TGF-alpha, and FGF7; ix) or from TGF-alpha and a FGF. In certainembodiments, the disclosure includes a stem cell culture mediacomprising a Wnt surrogate molecule disclosed herein, e.g., optionallyin combination with one or more of the growth factors or combinationsthereof described herein.

These methods of enhancing proliferation of stem cells can be used togrow new organoids and tissues from stem cells, as for example describedin WO2010/090513 WO02012/014076, Sato et al., 2011 (GASTROENTEROLOGY2011; 141: 1762-1772) and Sato et al., 2009 (Nature 459, 262-5).

In some embodiments, the Wnt surrogate molecules are used to enhancestem cell regeneration. Illustrative stem cells of interest include butare not limited to: muscle satellite cells; hematopoietic stem cells andprogenitor cells derived therefrom (U.S. Pat. No. 5,061,620); neuralstem cells (see Morrison et al. (1999) Cell 96: 737-749); embryonic stemcells: mesenchymal stem cells; mesodermal stem cells; liver stem cells;adipose-tissue derived stem cells, etc.

Other embodiments of the present invention relate, in part, todiagnostic applications for detecting the presence of cells or tissuesexpressing one or more Fzd receptors or LRP5 or LRP6 receptors. Thus,the present disclosure provides methods of detecting one or more Fzdreceptor or LRP5 or LRP6 receptor in a sample, such as detection ofcells or tissues expressing Fzd1. Such methods can be applied in avariety of known detection formats, including, but not limited toimmunohistochemistry (IHC), immunocytochemistry (ICC), in situhybridization (ISH), whole-mount in situ hybridization (WISH),fluorescent DNA in situ hybridization (FISH), flow cytometry, enzymeimmuno-assay (EIA), and enzyme linked immuno-assay (ELISA), e.g., bydetecting binding of a Wnt surrogate molecule.

ISH is a type of hybridization that uses a labeled complementary DNA orRNA strand (i.e., primary binding agent) to localize a specific DNA orRNA sequence in a portion or section of a cell or tissue (in situ), orif the tissue is small enough, the entire tissue (whole mount ISH). Onehaving ordinary skill in the art would appreciate that this is distinctfrom immunohistochemistry, which localizes proteins in tissue sectionsusing an antibody as a primary binding agent. DNA ISH can be used ongenomic DNA to determine the structure of chromosomes. Fluorescent DNAISH (FISH) can, for example, be used in medical diagnostics to assesschromosomal integrity. RNA ISH (hybridization histochemistry) is used tomeasure and localize mRNAs and other transcripts within tissue sectionsor whole mounts.

In various embodiments, the Wnt surrogate molecules described herein areconjugated to a detectable label that may be detected directly orindirectly. In this regard, an antibody “conjugate” refers to a Wntsurrogate molecule that is covalently linked to a detectable label. Inthe present invention, DNA probes, RNA probes, monoclonal antibodies,antigen-binding fragments thereof, and antibody derivatives thereof,such as a single-chain-variable-fragment antibody or an epitope taggedantibody, may all be covalently linked to a detectable label. In “directdetection”, only one detectable antibody is used, i.e., a primarydetectable antibody. Thus, direct detection means that the antibody thatis conjugated to a detectable label may be detected, per se, without theneed for the addition of a second antibody (secondary antibody).

A “detectable label” is a molecule or material that can produce adetectable (such as visually, electronically or otherwise) signal thatindicates the presence and/or concentration of the label in a sample.When conjugated to an antibody, the detectable label can be used tolocate and/or quantify the target to which the specific antibody isdirected. Thereby, the presence and/or concentration of the target in asample can be detected by detecting the signal produced by thedetectable label. A detectable label can be detected directly orindirectly, and several different detectable labels conjugated todifferent specific-antibodies can be used in combination to detect oneor more targets.

Examples of detectable labels, which may be detected directly, includefluorescent dyes and radioactive substances and metal particles. Incontrast, indirect detection requires the application of one or moreadditional antibodies, i.e., secondary antibodies, after application ofthe primary antibody. Thus, the detection is performed by the detectionof the binding of the secondary antibody or binding agent to the primarydetectable antibody. Examples of primary detectable binding agents orantibodies requiring addition of a secondary binding agent or antibodyinclude enzymatic detectable binding agents and hapten detectablebinding agents or antibodies.

In some embodiments, the detectable label is conjugated to a nucleicacid polymer which comprises the first binding agent (e.g., in an ISH,WISH, or FISH process). In other embodiments, the detectable label isconjugated to an antibody which comprises the first binding agent (e.g.,in an IHC process).

Examples of detectable labels which may be conjugated to Wnt surrogatemolecules used in the methods of the present disclosure includefluorescent labels, enzyme labels, radioisotopes, chemiluminescentlabels, electrochemiluminescent labels, bioluminescent labels, polymers,polymer particles, metal particles, haptens, and dyes.

Examples of fluorescent labels include 5-(and 6)-carboxyfluorescein, 5-or 6-carboxyfluorescein, 6-(fluorescein)-5-(and 6)-carboxamido hexanoicacid, fluorescein isothiocyanate, rhodamine, tetramethylrhodamine, anddyes such as Cy2, Cy3, and Cy5, optionally substituted coumarinincluding AMCA, PerCP, phycobiliproteins including R-phycoerythrin (RPE)and allophycoerythrin (APC), Texas Red, Princeton Red, green fluorescentprotein (GFP) and analogues thereof, and conjugates of R-phycoerythrinor allophycoerythrin, inorganic fluorescent labels such as particlesbased on semiconductor material like coated CdSe nanocrystallites.

Examples of polymer particle labels include micro particles or latexparticles of polystyrene, PMMA or silica, which can be embedded withfluorescent dyes, or polymer micelles or capsules which contain dyes,enzymes or substrates.

Examples of metal particle labels include gold particles and coated goldparticles, which can be converted by silver stains. Examples of haptensinclude DNP, fluorescein isothiocyanate (FITC), biotin, and digoxigenin.Examples of enzymatic labels include horseradish peroxidase (HRP),alkaline phosphatase (ALP or AP), β-galactosidase (GAL),glucose-6-phosphate dehydrogenase, β-N-acetylglucosamimidase,β-glucuronidase, invertase, Xanthine Oxidase, firefly luciferase andglucose oxidase (GO). Examples of commonly used substrates forhorseradishperoxidase include 3,3′-diaminobenzidine (DAB),diaminobenzidine with nickel enhancement, 3-amino-9-ethylcarbazole(AEC), Benzidine dihydrochloride (BDHC), Hanker-Yates reagent (HYR),Indophane blue (IB), tetramethylbenzidine (TMB), 4-chloro-1-naphtol(CN), .alpha.-naphtol pyronin (.alpha.-NP), o-dianisidine (OD),5-bromo-4-chloro-3-indolylphosphate (BCIP), Nitro blue tetrazolium(NBT), 2-(p-iodophenyl)-3-p-nitrophenyl-5-phenyl tetrazolium chloride(INT), tetranitro blue tetrazolium (TNBT),5-bromo-4-chloro-3-indoxyl-beta-D-galactoside/ferro-ferricyanide(BCIG/FF).

Examples of commonly used substrates for Alkaline Phosphatase includeNaphthol-AS-B 1-phosphate/fast red TR (NABP/FR),Naphthol-AS-MX-phosphate/fast red TR (NAMP/FR),Naphthol-AS-B1-phosphate/-fast red TR (NABP/FR),Naphthol-AS-MX-phosphate/fast red TR (NAMP/FR),Naphthol-AS-B1-phosphate/new fuschin (NABP/NF), bromochloroindolylphosphate/nitroblue tetrazolium (BCIP/NBT),5-Bromo-4-chloro-3-indolyl-b-d-galactopyranoside (BCIG).

Examples of luminescent labels include luminol, isoluminol, acridiniumesters, 1,2-dioxetanes and pyridopyridazines. Examples ofelectrochemiluminescent labels include ruthenium derivatives. Examplesof radioactive labels include radioactive isotopes of iodide, cobalt,selenium, tritium, carbon, sulfur and phosphorous.

Detectable labels may be linked to the antibodies described herein or toany other molecule that specifically binds to a biological marker ofinterest, e.g., an antibody, a nucleic acid probe, or a polymer.Furthermore, one of ordinary skill in the art would appreciate thatdetectable labels can also be conjugated to second, and/or third, and/orfourth, and/or fifth binding agents or antibodies, etc. Moreover, theskilled artisan would appreciate that each additional binding agent orantibody used to characterize a biological marker of interest may serveas a signal amplification step. The biological marker may be detectedvisually using, e.g., light microscopy, fluorescent microscopy, electronmicroscopy where the detectable substance is for example a dye, acolloidal gold particle, a luminescent reagent. Visually detectablesubstances bound to a biological marker may also be detected using aspectrophotometer. Where the detectable substance is a radioactiveisotope detection can be visually by autoradiography, or non-visuallyusing a scintillation counter. See, e.g., Larsson, 1988,Immunocytochemistry: Theory and Practice, (CRC Press, Boca Raton, Fla.);Methods in Molecular Biology, vol. 80 1998, John D. Pound (ed.) (HumanaPress, Totowa, N.J.).

The invention further provides kits for detecting one or more Fzd orLRP5/6 receptor or cells or tissues expressing one or more Fzd or LRP5/6receptors in a sample, wherein the kits contain at least one antibody,polypeptide, polynucleotide, vector or host cell as described herein. Incertain embodiments, a kit may comprise buffers, enzymes, labels,substrates, beads or other surfaces to which the antibodies of theinvention are attached, and the like, and instructions for use.

All of the above U.S. patents, U.S. patent application publications,U.S. patent applications, foreign patents, foreign patent applicationsand non-patent publications referred to in this specification and/orlisted in the Application Data Sheet, are incorporated herein byreference, in their entirety.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

EXAMPLES Example 1 Illustrative Wnt Surrogate Molecule Formats

Wnt surrogates having a variety of different formats disclosed hereinwere made. These included the following illustrative formats, eachcomprising a binding domain (“binder”) that binds to one or more Fzdreceptor and a binding domain (“binder”) that binds to an LRP5 and/orLRP6 receptor.

As shown in FIG. 1A, if a binder to one receptor is a Fab and to theother receptor is a Nab or scFv, they can be put together in severaldifferent configurations. In certain instances, the Fab binder first canbe reformatted to a full IgG format, then the Nab binder can be fused toany of the 4 available termini of the IgG. For example, the Nab can befused to the N-terminus of the IgG light chain (LC, the fusion will bereferred to as NL, shown in top left), N-terminus of the IgG heavy chain(HC, the fusion will be referred to as NH, shown in top right), theC-terminus of LC (the fusion will be referred to as CL, shown in middleright), and the C-terminus of HC (the fusion will be referred to as CH,shown in middle left). The linkers and the length of the linkers betweenthe IgG and the Nab can be varied. These four formats are bispecific andbivalent, they are bivalent binders toward each of the receptors. Analternative way to put the two binders together is the Hetero-Ig formatwhere the Fab binder is presented as a half antibody, and the Nab isfused to the N-terminus of an Fc (shown in lower middle). The two halvesmay be brought together by mutations in the CH3 domain (such as theknobs-into-holes) that favor the formation of the heterodimer. Thelinker and its length between the Nab binder and the Fc can be varied.This format would be bispecific but monovalent toward each receptor. TheNab part of any of the formats described in this example can also bereplaced by scFv fragments of binders as well.

As shown in FIG. 1B, if a binder to one receptor is a Fab and to theother receptor is also a Fab, they can be put together in severaldifferent configurations. In one approach, one Fab binder is firstreformatted to a full IgG format (shown in top). The second Fab bindercan be fused to the N-terminus of the IgG. The two HC can be fusedtogether with a linker in between. The LCs can be fused or unfused. Thelinker and its length can be varied. This format is a bispecific andbivalent format. Alternatively, the second Fab binder LC can be fused tothe HC of the IgG with a linker of various length in between. The secondFab binder HC can be fused or unfused to the LC of the IgG. A variationof this format has been called Fabs-in-tandem IgG (or FIT-Ig). Inanother approach, the two binders can be brought together as a Hetero-Igby mutations in the CH3 domain that favors the heterodimer assembly, thetwo arms will each bind to one receptor (shown in bottom). This formatis a bispecific and monovalent binder.

As shown in FIG. 1C, if a binder to one receptor is a Nab and to theother receptor is also a Nab, they can be put together in severaldifferent configurations. In the bispecific bivalent formats, in certaininstances, the two Nab binders can be fused together in tandem (shown intop row) or fused to the two-different ends of the Fc (shown in middlerow). The linker and its length between Nab and Nab or Nab and Fc can bevaried. Alternatively, the two Nabs can be assembled together asHetero-Ig to generate a bispecific and monovalent format (shown inbottom row). Similar to FIG. 1A, the Nab domains here can also bereplaced by an scFv domain of a binder. In all the examples, the Nab andscFv can be mixed in certain combinations as well.

As shown in FIG. 1D, the binders against Fzd and LRP can also be linkedtogether in a diabody (or DART) configuration. The diabody can also bein a single chain configuration. If the diabody is fused to an Fc, thiswill create a bivalent bispecific format. Without fusion to Fc, thiswould be a monovalent bispecific format.

A number of Wnt surrogates representing different configurations wereproduced. These included the Wnt surrogates described in Table 3. Theseillustrative Wnt surrogates include one, two or three polypeptides, thesequences of which are provided as Sequence 1, Sequence 2, and/orSequence 3. The sequence may include a leader peptide sequence, a Nabsequence, a linker, and/or a heavy or light chain sequence. Annotatedsequences are provided in FIG. 19, in which the leader peptide sequenceis italicized, the linker sequence is underlined, the Nab sequence isshown in bold, and the remaining sequence is the heavy chain or lightchain sequence. The Fzd binder IDs and LRP binder IDs correspond to theclone numbers provided in Tables 1A-B and 2A-B for various Fzd bindingor LRP5/6 binding antibodies or antigen-binding fragments thereof.

The Wnt surrogates beginning with “R2M3” include different LRP6 bindingdomains fused to the N-terminus of a light chain region of the anti-Fzdantibody or antigen-binding fragment thereof named 001S-A04. The firstsix Wnt surrogates beginning with “18R5” in Table 3 include differentLRP6 binding domains fused to the N-terminus of the anti-Fzd antibody orantigen-binding fragment thereof named 18R5. The Wnt surrogatesbeginning with “1R” include the anti-LRP6 antibody of antigen-bindingfragment thereof named “009S-E04” fused to the N-terminus of differentanti-Fzd antibodies or antigen-binding fragment thereof. For“R2M3-26CH,” the LRP6 binding region is fused to the C-terminus of theFzd binding region. For “R2M3-26NH,” the LRP6 binding region is fused tothe N-terminus of the Fzd binding region. For “R2M3-26CL,” the LRP6binding region is fused to the C-terminus of the Fzd binding region. For“R2M3-26NL,” the LRP6 binding region is fused to the N-terminus of theFzd binding region. For “R2M3-26Fab” and “R2M3-32Fab,” the LRP6 bindingregion is fused to the N-terminus of the Fzd binding region. For“Hetero-Ig,” the LRP6 binding region is fused to the N-terminus of humanFc_hole, and paired with Fzd binder light chain and heavy chain humanIgG1_knob. The Wnt surrogates beginning with “17SB9” include differentLRP6 binding domains fused to the N-terminus of a light chain region ofthe anti-Fzd antibody or antigen-binding fragment thereof named017S-B09. The Wnt surrogates beginning with “1R-C07” include differentLRP6 binding domains fused to the N-terminus of a light chain region ofthe anti-Fzd antibody or antigen-binding fragment thereof named001S-B03. The Wnt surrogates beginning with “R2M13” include differentLRP6 binding domains fused to the N-terminus of a light chain region ofthe anti-Fzd antibody or antigen-binding fragment thereof named004S-G06. The Wnt surrogates beginning with “3SD10” include differentLRP6 binding domains fused to the N-terminus of a light chain region ofthe anti-Fzd antibody or antigen-binding fragment thereof named003S-D10. The Wnt surrogates beginning with “4SD1” include differentLRP6 binding domains fused to the N-terminus of a light chain region ofthe anti-Fzd antibody or antigen-binding fragment thereof named004S-D01. The Wnt surrogates beginning with “14SB6” include differentLRP6 binding domains fused to the N-terminus of a light chain region ofthe anti-Fzd antibody or antigen-binding fragment thereof named014S-B06.

TABLE 3 Wnt Surrogate Sequences Fzd LRP Sequence 1 Sequence 2 Sequence 3Name binder ID binder ID SEQ ID NO SEQ ID NO SEQ ID NO R2M3-23 001S-A04009S-B04 89 95 N/A R2M3-26 001S-A04 009S-E04 90 95 N/A R2M3-28 001S-A04009S-G04 91 95 N/A R2M3-29 001S-A04 009S-H04 92 95 N/A R2M3-31 001S-A04013S-G04 93 95 N/A R2M3-32 001S-A04 013S-H04 94 95 N/A 18R5-5 18R5008S-G01 96 104 N/A 18R5-7 18R5 008S-C02 97 104 N/A 18R5-8 18R5 008S-D0298 104 N/A 18R5-9 18R5 008S-E02 99 104 N/A 18R5-26 18R5 009S-E04 100 104N/A 18R5-28 18R5 009S-G04 101 104 N/A 18R5-31 18R5 013S-G04 102 104 N/A18R5-32 18R5 013S-H04 103 104 N/A 1R-B05-26 001S-E02 009S-E04 105 111N/A 1R-C01-26 001S-B01 009S-E04 106 112 N/A 1R-C07-26 001S-B03 009S-E04107 113 N/A 1R-E06-26 001S-H02 009S-E04 108 114 N/A 1R-G05-26 001S-G02009S-E04 109 115 N/A 1R-G06-26 001S-A03 009S-E04 110 116 N/A R2M3-26CH001S-A04 009S-E04 125 117 N/A R2M3-26NH 001S-A04 009S-E04 125 118 N/AR2M3-26CL 001S-A04 009S-E04 119 2254 N/A R2M3-26NL 001S-A04 009S-E04 1202254 N/A R2M3-26Fab 001S-A04 009S-E04 120 122 N/A R2M3-26F(ab’)2001S-A04 009S-E04 120 2252 R2M3-32Fab 001S-A04 013S-H04 123 122 N/AR2M3-26Hetero-Ig 001S-A04 009S-E04 125 126 127 26-17SB9 017S-B09009S-E04 128 N/A N/A 26:Fc:17SB9-criss-cross 017S-B09 009S-E04 2192 2193N/A 26:5:17SB9:Fc 017S-B09 009S-E04 2194 N/A N/A 26:10:17SB9:Fc 017S-B09009S-E04 2195 N/A N/A 26:15:17SB9: Fc 017S-B09 009S-E04 2196 N/A N/A17SB9:Fc:26 017S-B09 009S-E04 2197 N/A N/A 26:Fc:26+17SB9:Fc:17SB9017S-B09 009S-E04 2198 2199 N/A 1R-C07-3 001S-B03 008S-D01 134 113 N/A1R-C07-36 001S-B03 013S-D05 135 113 N/A R2M13-3 004S-G06 008S-D01 136153 N/A R2M13-26 004S-G06 009S-E04 137 153 N/A R2M13-36 004S-G06013S-D05 138 153 N/A R2M3-3 001S-A04 008S-D01 139 2254 N/A R2M3-36001S-A04 013S-D05 140 2254 N/A 3SD10-3 003S-D10 008S-D01 141 154 N/A3SD10-26 003S-D10 009S-E04 142 154 N/A 3SD10-36 003S-D10 013S-D05 143154 N/A 4SD1-3 004S-D01 008S-D01 144 155 N/A 4SD1-26 004S-D01 009S-E04145 155 N/A 4SD1-36 004S-D01 013S-D05 146 155 N/A 14SB6-3 014S-B06008S-D01 147 156 N/A 14SB6-26 014S-B06 009S-E04 148 156 N/A 14SB6-36014S-B06 013S-D05 149 156 N/A R2M9-3 003S-E07 008S-D01 150 157 N/AR2M9-26 003S-E07 009S-E04 151 157 N/A R2M9-36 003S-E07 013S-D05 152 157N/A 18R5:5:1115.3:Fc 18R5 1115.3 2200 N/A N/A 18R5:10:1115.3:Fc 18R51115.3 2201 N/A N/A 18R5:15:1115.3:Fc 18R5 1115.3 2202 N/A N/A1115.3:5:18R5:Fc 18R5 1115.3 2203 N/A N/A 1115.3:10:18R5:Fc 18R5 1115.32204 N/A N/A 1115.3:15:18R5:Fc 18R5 1115.3 2205 N/A N/A18R5:5:YW211.31.57:Fc 18R5 YW211.31.57 2206 N/A N/A18R5:10:YW211.31.57:Fc 18R5 YW211.31.57 2207 N/A N/A18R5:15:YW211.31.57:Fc 18R5 YW211.31.57 2208 N/A N/AYW211.31.57:5:18R5:Fc 18R5 YW211.31.57 2209 N/A N/AYW211.31.57:10:18R5:Fc 18R5 YW211.31.57 2210 N/A N/AYW211.31.57:15:18R5:Fc 18R5 YW211.31.57 2211 N/A N/A 18R5:Fc:1115.3 18R51115.3 2212 N/A N/A 1115.3:Fc:18R5 18R5 1115.3 2213 N/A N/A18R5:Fc:YW211.31.57 18R5 YW211.31.57 2250 N/A N/A YW211.31.57:Fc:18R518R5 YW211.31.57 2267 N/A N/A 421.1-R2M3 cp 001S-A04 421.1 2214 22152216 1RC07:5:10SA7 cp 1RC07 10SA7 2217 2218 2219 1RC07:10:10SA7 cp 1RC0710SA7 2217 2218 2222 1RC07:15:10SA7 cp 1RC07 10SA7 2217 2218 22251RC07:5:10SG7 cp 1RC07 10SG7 2217 2227 2228 1RC07:10:10SG7 cp 1RC0710SG7 2217 2227 2231 1RC07:15:10SG7 cp 1RC07 10SG7 2217 2227 223410SG7:5:1RC07 cp 1RC07 10SG7 2227 2217 2237 10SG7:10:1RC07 cp 1RC0710SG7 2227 2217 2240 10SG7:15:1RC07 cp 1RC07 10SG7 2227 2217 22431RC07:5:10SA7 L->H 1RC07 10SA7 2244 2245 2246 1115.3:5:R2M3 L->H001S-A04 1115.3 2247 125 2248 1115.3:10:R2M3 L->H 001S-A04 1115.3 2247125 2249 10SG11-1RC07 1RC07 10SG11 2252 2253 N/A 18R5:5:1115.3:His 18R51115.3 2255 N/A N/A 18R5:10:1115.3:His 18R5 1115.3 2256 N/A N/A18R5:15:1115.3:His 18R5 1115.3 2257 N/A N/A 1115.3:5:18R5:His 18R51115.3 2258 N/A N/A 1115.3:10:18R5:His 18R5 1115.3 2259 N/A N/A1115.3:15:18R5:His 18R5 1115.3 2260 N/A N/A 18R5:5:YW211.31.57:His 18R5YW211.31.57 2261 N/A N/A 18R5:10:YW211.31.57:His 18R5 YW211.31.57 2262N/A N/A 18R5:15:YW211.31.57:His 18R5 YW211.31.57 2263 N/A N/AYW211.31.57:5:18R5:His 18R5 YW211.31.57 2264 N/A N/AYW211.31.57:10:18R5:His 18R5 YW211.31.57 2265 N/A N/AYW211.31.57:15:18R5:His 18R5 YW211.31.57 2266 N/A N/A

Example 2 Characterization of a Wnt Surrogate Molecule, R2M3-26

The R2M3-26 molecule consists of a Fzd binder (R2M3) and a LRP6 binder(26). The LRP6 binder 26 was fused to the N-terminus of R2M3 LC with a5-amino acid linker as depicted in FIG. 2A. R2M3 was in the form of anIgG. The protein was purified by Protein A affinity column followed by asize-exclusion-chromatography (SEC) step. The absorbance trace from theSEC and the SDS-PAGE gels of the SEC fractions were shown in FIG. 2B.The ability of R2M3-26 to activate canonical Wnt signaling was tested ina Wnt responding 293 reporter cell line (293STF). The 293STF reporteractivity traces across the SEC fractions were shown in FIG. 2B, the peakof the reporter activity correlated with the peak of the proteins. Thepeak fraction was further characterized by a dose response in 293STFcells in the absence and presence of R-spondin (FIG. 2D). R2M3-26induced reporter activity in a dose dependent manner and was enhanced bythe presence of R-spondin similar to a natural Wnt ligand, while R2M3IgG alone without the attachment of the LRP binding arm did not inducereporter activity. The ability of R2M3-26 to interact with its target, aFzd1 ECD, was performed in Octet interaction assay (FIG. 2C), and theresults showed that the fusion of the LRP6 binding arm, 26, did notaffect R2M3 interaction with its target, Fzd.

Example 3 Characterization of a Wnt Surrogate Molecule, R2M3-32

The R2M3-32 molecule consists of a Fzd binder (R2M3) and a LRP6 binder(32). The LRP6 binder 32 was fused to the N-terminus of R2M3 LC with a5-amino acid linker as depicted in FIG. 3A. R2M3 was in the form of anIgG. The protein was purified by Protein A affinity column followed by asize-exclusion-chromatography (SEC) step. The absorbance trace from theSEC and the SDS-PAGE gels of the SEC fractions were shown in FIG. 3B.The ability of R2M3-32 to activate canonical Wnt signaling was tested ina Wnt responding 293 reporter cell line (293STF). The 293STF reporteractivity traces across the SEC fractions were shown in FIG. 3B, the peakof the reporter activity correlated with the peak of the proteins. Thepeak fraction was further characterized by a dose response in 293STFcells in the absence and presence of R-spondin (FIG. 3D). R2M3-32induced reporter activity in a dose dependent manner and was enhanced bythe presence of R-spondin, while R2M3 IgG alone without the attachmentof the LRP binding arm did not induce reporter activity. The ability ofR2M3-32 to interact with its target, a Fzd1 extracellular domain (ECD),was performed in Octet interaction assay (FIG. 3C). The results showedthat the fusion of the LRP6 binding arm, 32, did not affect R2M3interaction with its target, Fzd.

Example 4 R2M3-26 and R2M3-32 Activities can be Inhibited by Soluble FzdECD and by R2M3 IgG Alone without the LRP Binding Arm

The ability of soluble Fzd extracellular domain (ECD) or R2M3 IgG aloneto inhibit Wnt surrogates was determined using the 293STF reporterassay. The Fzd1 ECD-Fc or R2M3 IgG was titrated into the 293STF reporterassay, at a fixed concentration of R2M3-26 or R2M3-32. In a dosedependent manner, both Fzd1 ECD-Fc and R2M3 IgG inhibited R2M3-26 (FIG.4A) and R2M3-32 (FIG. 4B) induced reporter signaling, while the negativecontrol molecule, Fc alone had no impact.

Example 5 Characterization of R2M3-LRP6 Binder Fusions in 293, Huh7,A375, BNL.CL2 Wnt Dependent Reporter Assays

The Fzd binder, R2M3, was fused to additional LRP6 binders, 23, 25, 26,27, 28, 29, 31, 32, 33, and 36. The LRP6 binders were Nab and were fusedto the N-terminus of R2M3 LC with a 5-amino acid linker. These proteinswere purified by Protein A affinity column followed by a SEC step. Thefusion proteins were tested in Wnt dependent reporter assays in 293,Huh7, A375, and BNL.CL2 cell lines, and activated Wnt signaling tovarious levels. R2M3 was also fused to two non-LRP6 binder Nabs, 24 and34, in the same format as the Lrp6 binders. These two non-bindersdisplayed no activity in Wnt dependent 293 reporter assay (FIG. 5),suggesting that Wnt activities observed with R2M3 fusions to 23, 25, 26,27, 28, 29, 31, 32, 33, and 36 are dependent on the presence of both Fzdand Lrp mimicking the nature ligand function.

Example 6 Characterization of 18R5-LRP6 Binder Fusions in 293, A375, andBNL.CL2 Wnt Dependent Reporter Assays

The Fzd binder, 18R5, was fused to LRP6 binders, 26, 28, 31, 32. TheLRP6 binders were Nab and were fused to the N-terminus of 18R5 LC with a5-amino acid linker. These proteins were purified by Protein A affinitycolumn followed by a SEC step. The fusion proteins were tested in Wntdependent reporter assays in 293, A375, and BNL.CL2 cell lines anddemonstrated ability to activate Wnt signaling (FIG. 6).

Example 7 Characterization of 18R5-LRP5 Binder Fusions in 293 WntDependent Reporter Assays

The Fzd binder, 18R5, was fused to LRP5 binders, 5, 7, 8, 9. The LRP5binders were Nab and were fused to the N-terminus of 18R5 LC with a5-amino acid linker. These proteins were purified by Protein A affinitycolumn followed by a SEC step. The fusion proteins were tested in a Wntdependent reporter assays in 293 cells and were able to activate Wntsignaling (FIG. 7).

Example 8 Characterization of Various Fzd Binders-LRP6 Binder 26 Fusionsin 293 Wnt Dependent Reporter Assays

The various Fzd binders, 1R-B05, 1R-C01, 1R-C07, 1R-E01, 1R-E06, 1R-G05,1R-G06, 1R-H04, in IgG format were fused to LRP6 binders, 26. The LRP6binder Nab was fused to the N-terminus of various Fzd binder LC with a5-amino acid linker. These proteins were purified by Protein A affinitycolumn followed by a SEC step. The SDS-PAGE gel analysis of the SEC peakfractions were shown in FIG. 8A. The fusion proteins were tested in aWnt dependent reporter assays in 293 cells in the presence of Rspo andwere able to activate Wnt signaling (FIG. 8B).

Example 9 SAR Analysis of the IgG-Nab Fusion Format

SAR analysis of the IgG-Nab fusions was performed by rotating theattachment location of the Nab to the different termini of the IgG HC orLC as depicted in FIG. 1A. CH indicates attaching the Nab to theC-terminus of heavy chain; NH indicates attaching the Nab to theN-terminus of heavy chain; CL indicates attaching the Nab to theC-terminus of light chain; NL indicates attaching the Nab to theN-terminus of light chain. Three pairs of IgG-Nab fusions SARs wereshown, the pairs were between R2M3 and 26, between R2M3 and 32, andbetween 18R5 and 26. The assays were performed on Wnt responsive 293reporter cells in the presence of Rspo and activated Wnt signaling tovarious levels (FIG. 9). These results demonstrate that the attachmentlocation of the fusion and the geometry between the Fzd and LRP bindingdomains play roles in the ability of the Wnt surrogates to activate Wntsignaling.

Example 10 Characterization of R2M3-26 in the Fab Format

The molecule R2M3-26 Fab consists of a Fzd binder (R2M3) and a LRP6binder (26). The LRP6 binder 26 was fused to the N-terminus of R2M3 LCwith a 5-amino acid linker as depicted in FIG. 10A. R2M3 was in the formof a Fab. The protein was purified by Ni-NTA affinity column followed bya size-exclusion-chromatography (SEC) step. The absorbance trace fromthe SEC and the SDS-PAGE gels of the SEC fractions are shown in FIG.10B. The ability of R2M3-26 as a Fab format to activate canonical Wntsignaling was tested in a Wnt responding 293 reporter cells (293STF).The 293STF reporter activity traces across the SEC fractions are shownin FIG. 10B. Unlike when R2M3 was in the IgG format shown in FIG. 2, thepeak of the reporter activity from the R2M3 in the Fab format did notcorrelate with the peak of the proteins. These results suggest thatR2M3-26 fusion in the Fab format is ineffective in inducing canonicalWnt signaling as detected by a reporter assay.

Example 11 Characterization of R2M3-32 in the Fab Format

The molecule R2M3-32 Fab consists of a Fzd binder (R2M3) and a LRP6binder (32). The Lrp6 binder 32 was fused to the N-terminus of R2M3 LCwith a 5-amino acid linker as depicted in FIG. 11A. R2M3 was in the formof a Fab. The protein was purified by Ni-NTA affinity column followed bya size-exclusion-chromatography (SEC) step. The absorbance trace fromthe SEC and the SDS-PAGE gels of the SEC fractions were shown in FIG.11B. The ability of R2M3-32 as a Fab format to activate canonical Wntsignaling was tested in a Wnt responding 293 reporter cells (293STF).The 293STF reporter activity traces across the SEC fractions are shownin FIG. 11B. Unlike when R2M3 was in the IgG format shown in FIG. 3, thepeak of the reporter activity from the R2M3 in the Fab format did notcorrelate with the peak of the proteins. These results suggest thatR2M3-32 fusion in the Fab format is ineffective in inducing canonicalWnt signaling as detected by a reporter assay.

Example 12 Characterization of R2M3-26 in the Heterolg Format

The molecule R2M3-26 Heterolg consists of a Fzd binder (R2M3) and a LRP6binder (26) as depicted in FIG. 12A and described in FIG. 1A. Theprotein was purified by Protein A affinity column followed by asize-exclusion-chromatography (SEC) step. The peak fraction from the SECcolumn was tested in a dose response in the Wnt responsive 293STFreporter cells in the absence or presence of R-spondin (FIG. 12B).Compared to R2M3-26 in the IgG format (as described in FIG. 2), R2M3-26Heterolg was ineffective in inducing canonical Wnt signaling as detectedin the 293 reporter assay.

Example 13 Characterization of 26-17SB9 in the Nab-Nab Format

The molecule 26-17SB9 Nab-Fc-Nab consists of a LRP6 binder (26) and aFzd binder (17SB9) as depicted in FIG. 13A and described in FIG. 1C. Theprotein was purified by Protein A affinity column followed by asize-exclusion-chromatography (SEC) step. The peak fraction from the SECcolumn was tested in a dose response in the Wnt responsive 293STFreporter cells in the absence or presence of R-spondin (FIG. 13B).26-17SB9 in the Nab-Fc-Nab format induced canonical Wnt signaling asdetected in the 293 reporter assay.

Additional combinations of 26 and 17SB9 were also constructed (FIG. 13C)and tested in 293 reporter assays. As shown in FIGS. 13D and 13E, thesevarious combinations where 26 and 17SB9 were arranged in differenttandem formats or on different ends of the Fc fragment all activated Wntsignaling to various levels in the presence of 20 nM R-spondin

Example 14 Characterization of 18R5-LRP6 Binder Fusions in Tandem scFvFormats

The Fzd binder 18R5, the LRP6E1E2 binder 1115.3 (as described in PCTPublication WO2009/064944), and the LRP6E3E4 binder YW211.31.57 (asdescribed in PCT Publication WO2011/119661) were converted into scFvformat. 1115.3_scFv or YW211.31.57_scFv is assembled to the N-terminusof 18R5_scFv with a 5, 10 or 15-amino acid linker and 18R5_scFvC-terminus is fused to a human Fc domain. In another set of examples,1115.3_scFv or YW211.31.57_scFv is assembled to the C-terminus of 18R5scFv with a 5, 10 or 15-amino acid linker and human Fc domain is fusedto the C-terminus of LRP binders. These formats are depicted in FIG. 14Gleft panel. In another example, 18R5_scFv and LRP binder, 1115.3_scFv,or LRP binder, YW211.31.57_scFv were fused to the two ends of a human Fcdomain (as depicted in FIG. 14G right panel). These proteins werepurified by Protein A affinity column followed by a SEC step.

The fusion proteins were tested in a Wnt dependent reporter assays in293 cells. 18R5_scFv-1115.3_scFv-Fc and 1115.3_scFv-18R5_scFv-Fc with 5,10 or 15-mer linker were able to activate Wnt signaling (FIGS. 14A and14B). 18R5_scFv-YW211.31.57_scFv-Fc and YW211.31.57_scFv-18R5_scFv-Fcwith different linkers activated Wnt signaling (FIGS. 14C and 14D). Inaddition, 18R5_scFv and 1115.3_scFv or YW211.31.57 fused to the two endsof Fc also activated Wnt signaling (FIGS. 14E and F). While all of thesescFv formats activated Wnt signaling, potency and overall maximalefficacy may differ depends on the binder combination, linker length,and relative orientation.

In another example, 1115.3_scFv or YW211.31.57_scFv is assembled to theN-terminus or C-terminus of 18R5_scFv with a 5, 10 or 15-amino acidlinker without the further fusion of Fc, to create a bispecific butmonovalent binding to each of Fzd or LRP. As shown in FIG. 14H, the1115.3_scFv and 18R5_scFv fusions were in effective in activating Wntsignaling in the presence of 20 nM R-spondin in 293 reporter cells.

Example 15 Generation of Wnt Surrogate Molecules in the Fab-IgG Format

Wnt mimetic or surrogate molecules can be generated in various formatswhen both the FZD and LRP binders are Fabs. Various approaches, such ascharge paring, “knobs-in-holes”, crossover of the heavy and light chainsof Fabs, can be employed to ensure proper heavy and light chain pairing.Two examples are given below.

-   -   1. Charge-pairing (cp) approach for Fab-on-IgG format: The heavy        chain (VH-CH1) domain of an anti-LRP6 Fab, through a linker of        5, 10, or 15-mer amino acids, was fused in tandem with the        N-terminus of the heavy chain (VH-CH1-CH2-CH3) of an anti-FZD        binder. Both VH-CH1 domains of anti-LRP6 and anti-FZD contain        three amino acid mutations (Q39D, Q105D, S183K in the anti-LRP6        Fab; Q39K, Q105K, S183E in anti-FZD Fab) each for proper paring        with their own partner light chains, which also contain three        complementary amino acid mutations (Q38K, A/S43K, S176E in        anti-LRP6 light chain; Q38D, A/S43D, S176K in the anti-FZD light        chain). The order of the anti-LRP6 and anti-FZD Fabs could be        reversed, where the anti-FZD binder is a Fab and is fused to        anti-LRP binder which is in IgG format (FIG. 15A).    -   2. HC-LC cross over approach for Fab-on-IgG format: The light        chain (VL-CL) domains of anti-LRP6 binder was, through a linker        of 5, 10, or 15-mer amino acids, fused in tandem with the        N-terminus of the heavy chain (VH-CH1-CH2-CH3) of an anti-FZD        binder. The second construct was VH-CH1 of the anti-LRP6 binder        and the third construct was VL-CL of the anti-FZD binder.        Similar to the example above, the order of the anti-LRP6 and the        anti-FZD binders could be reversed, where anti-FZD binder Fab is        fused to the N-terminus of the anti-LRP binder which is in IgG        format (FIG. 15A).

Several different pairs of LRP and FZD binders were assembled in theseformats and tested in a Wnt responding 293 reporter cell line (293STF).As an example, the anti LRP6E1E2 binder 421.1 (as described in PCTPublication WO2009/064944) was fused to the N-terminus of anti-FZDbinder, R2M3, using the charge paring approach to generate, 421.1-R2M3cp. 421.1-R2M3 cp dose-dependently activated Wnt signaling in the 293reporter assay (FIG. 15B). An anti-FZD binder, 1RC07, was fused to theN-terminus of an anti-LRP binder, 10SA7, with 5, 10, or 15-mer linkers.All three fusion proteins activated Wnt signaling (FIG. 15C). Theanti-FZD binder, 1RC07, was further fused with the anti-LRP binder,10SG7, either with 1RC07 in the Fab format fused to the N-terminus of10SG7 in IgG format, or in the reverse order where 10SG7 as Fab fused tothe N-terminus of 1RC07 as IgG with either 5, 10, or 15-mer amino acidlinkers. All fusion molecules activated Wnt signaling while somepreference of orientation and linker length was observed (FIGS. 15D and15E).

The HC-LC crossover Fab-IgG format was also tested. The anti-FZD binder1RC07 LC was fused to the N-terminus of the anti-LRP6 binder 10SA7 HC togenerate 1RC07-5: 10SA7 L->H. The LC of anti-LRP6E1E2 binder 1115.3 (asdescribed in PCT Publication WO2009/064944) was fused to the N-terminusof the anti-FZD binder R2M3 HC with 5 or 10-mer linkers to generate1115.3:5:R2M3 L->H or 1115.3:10:R2M3 L->H, respectively. These moleculesalso activated Wnt signaling (FIGS. 15F and 15G).

Example 16 Characterization of R2M3-26 in the F(ab′)2 Format

R2M3-26 IgG1 was digested by IDES (Promega, WI) at 37° C. for 2 hours.Vast majority of the digested product was R2M3-26F(ab′)2 (FIG. 16A),some partially digested product with one Fab still attached to Fc(designated as R2M3-26F(ab′)2-Fc here) was also detected, and nouncleaved R2M3-26 was detected. The cleaved product was purified byanti-Lambda resin to remove the Fc fragment, then a SEC polishing wasfollowed to separate the R2M3-26F(ab′)2 from R2M3-26F(ab′)2-Fc. TheSDS-PAGE gel of the final purified protein is shown in FIG. 16B. TheR2M3-26F(ab′)2 activity was measured in STF assay in HEK293 cells.R2M3-26F(ab′)2 was able to activate Wnt signaling (FIG. 16C).

Example 17 Characterization of Additional Wnt Surrogate Molecules

The FZD binders were fused to LRP binders. The LRP5 or 6 binders wereNabs (or VHHs) in this example and were fused to the N-terminus of FZDbinders LC with a 5-amino acid linker (as represented in FIG. 17A).These proteins were purified by Protein A affinity column followed by aSEC step. The purified proteins were tested in Wnt dependent reporterassay in 293 cells (FIG. 17B, C, D, H), or 293 cells co-transfected withFZD4 expression construct (FIG. 17E, F), or 293 cells co-transfectedwith FZD9 expression construct (FIG. 17G) in the presence of 20 nMR-spondin. These molecules activated Wnt signaling with varying levelsof potency and efficacy.

Example 18 Characterization of 10SG11-1RC07 in the 2Fv-Ig Format

The molecule 10SG11-1RC07 consists of an N-terminal LRP binder (10SG11)and a Fzd binder (1RC07). The Fv of 10SG11 was fused to the N-terminusof 1RC07 with a 5-amino acid linker as depicted in FIG. 18A. 1RC07 wasin the form of an IgG1 with the Fc mutations L234A/L235A/P329G. Theprotein was purified by a Protein A affinity column followed by an SECstep. The fusion protein was tested in Wnt dependent reporter assays in293 cell lines and demonstrated the ability to activate Wnt signaling(FIG. 18B-C)

Example 19 In Vivo PK/PD Characterization of R2M3-26

Six-week old C57BI/6J male mice were obtained from Jackson Laboratories(Bar Harbor, Me., USA) and were housed 3 per cage. All animalexperimentation was in accordance with the criteria of the “Guide forthe Care and Use of Laboratory Animals” prepared by the National Academyof Sciences. Protocols for animal experimentation were approved by theSurrozen Institutional Animal Care and Use Committee. Mice wereacclimatized a minimum of two days prior to initiating experiments. Micehad unlimited access to purified, laboratory-grade acidified water andwere fed ad libitum (2018 Teklad global 18% protein rodent diet) Micewere kept 12/12-hour light/dark cycle in a 30% to 70% humidityenvironment and room temperature ranging from 20° C. to 26° C.

For the pharmacokinetic (PK) study (FIG. 20A), n=3 per group was used.Mice were dosed with R2M3-26 (with effectorless Fc mutations) at 1 mg/kg(10 ml/kg in saline) either using intravenous (IV) or intraperitoneal(i.p.) injections. Mice were anesthetized with isoflurane and blood wasremoved from the retro-orbital plexus, tail vein or heart at 10 minutes,30 minutes, 1, 4, 24, 72 or 144 hours after injection. Blood was allowedto coagulate at room temperature, followed by centrifugation for 7minutes at 8,000 g. The serum was removed and stored at −20° C. untilthe measurement of serum R2M3-26 concentrations by ELISA with Anti HumanIgG Fc Fragment (Jackson Immuno Research Labs NC9747692).

For the pharmacodynamic (PD) study (FIG. 20B), n=6 per group was used.Mice were injected i.p. with R2M3-26 at the indicated doses (10 ml/kg insaline). Control mice received saline only. Eight hours later, mice wereanesthetized with isoflurane and the blood was collected by cardiacpuncture. Blood was allowed to coagulate at room temperature, followedby centrifugation for 7 minutes at 10,000 g. The serum was removed andstored at −20° C. until the measurement of serum R2M3-26 concentrationsby ELISA. A portion of the left liver lobe was snap-frozen in liquidnitrogen and stored at −80° C. for RNA analysis. RNA was extracted fromliver samples using the MagMAX™ mirVana™ Total RNA Isolation Kit(ThermoFisher, A27828). cDNA was produced using the high-Capacity cDNAReverse Transcription Kit (ThermoFisher, 43-688-14). Axin2 mRNAexpression was measured by using TaqMan® Fast Advanced Master Mix(ThermoFisher, 4444963) and the Mm00443610_m1 Axin2 Probe (Thermofisher,4331182).

These studies showed that R2M3-26 was stable, highly bioavailable andactive in vivo, as shown by the induction of Axin2 mRNA expression.

Example 20 In Vivo Bone Model and Characterization of AAV-Delivered WntSurrogates

In vivo experiments were conducted by infecting mice with an AAV vectorthat expressed Flag- and His-tagged 18R5-DKK1c protein(AAV-18R5-DKK1c-FlagHis). 18R5-DKK1c is a fusion protein containing thefrizzled binding antibody, 18R5, in scFv format, fused to DKK1c, asdescribed in PCT Publication WO2016/040895, e.g., FIG. 5. Control micewere treated with vehicle only, romosozumab, an AAV vector thatexpressed green fluorescent protein (GFP) (AAV-CAG-GFP), or an AAVvector that expressed a fusion protein comprising an anti-GFP scFv fusedto a mutant DKK1c (AAV-ScFv (anti-GFP)-DKK1cF234K-Flag-His). 28 daysafter infection, animals were sacrificed and bone mineral density, bonevolume and other characteristics were measured. As shown in FIGS.21A-21E, systemic expression of 18R5-DKK1c resulted in significantlyincreased bone mineral density as early as 14 days of 18R5-DKK1csystemic expression, as determined by dual X-ray absorptiometry (DEXA)scan. Systemic expression of 18R5-DKK1c increased bone mineral density(BMD) as measured by DEXA scan (FIG. 21A) and increased levels of theserum P1NP bone formation marker in naïve mice (FIG. 21C). Serum levelsof AAV-ScFv (anti-GFP)-DKK1cF234K and 18R5-DKK1c were detected in theserum and found to be well above the in vitro determined EC50 (FIG.21B). AAV-CAG-GFP and AAV-ScFv (anti-GFP)-DKK1cF234K were negativecontrols. Romosozumab was a positive control, and vehicle only was anegative control. 18R5-DKK1c also increase bone density in lumbarvertebra and whole body, as shown in FIGS. 20D and 20E, wherein *indicates P value <0.05 and ** indicates P value <0.0001. 18R5-DKK1cexpression through AAV also increased bone volume in tibia and femur andcortical thickness in femur mid-diaphysis at 28 days after treatment innaïve mice as measured by micro CT, as shown in FIGS. 22A-22D, wherein**** indicates P value <0.0001.

Systemic expression of 18R5-DKK1c resulted in significantly increasedmineral apposition rate from baseline to single label in last 8 days, asshown in FIGS. 23A and 23B.

Systemic expression of 18R5-DKK1c also resulted in increased osteoblastnumbers and decreased osteoclast numbers, as shown in FIGS. 24A-24D.

18R5-DKK1c treatment increased bone stiffness and ultimate load tofracture in biomechanical testing, suggesting improved resistance tofracture, as shown in FIGS. 25A-25C.

These studies demonstrated that systemic expression of 18R5-DKK1c usingAAV increased bone mineral density (BMD) as measured by DEXA, and alsoshowed that 18R5-DKK1c increased bone volume as measured by micro CT asearly as 14 days of treatment. 18R5-DKK1c also increased corticalthickness 28 days after treatment. Systemic expression of 18R5-DKK1cresulted in significantly increased mineral apposition rate, increasedosteoblast numbers, and decreased osteoclast numbers. It also increasedbone stiffness and ultimate load to fracture, suggesting improvedresistance to fracture.

Example 21 In Vivo Bone Model and Characterization of Wnt SurrogatesProduced as Recombinant Proteins

In vivo experiments were conducted by treating mice with recombinantlyproduced R2M3-26 protein at various dosages via i.p. injection. Controlmice were treated with vehicle only (negative control), romosozumab(positive control), anti-Beta-Galactosidase (negative control), orIgG2-anti-GFP (negative control). Bone mineral density (BMD) measured byDEXA and bone volume measured by micro CT were monitored longitudinallyat indicated time points. Four weeks after treatment, animals weresacrificed and bone characteristics were measured. For single injectionof R2M3-26, the experimental data was monitored and is shown for twoweeks after treatment.

Treatment with recombinant R2M3-26 induced rapid and sustained increaseof bone mineral density (BMD) and bone volume in naive mice, as shown inFIGS. 26A-26D. Both bone volume and BMD increased rapidly, suggestingresistance to fracture.

The ovariectomy induced osteoporosis model is a well-establishedhigh-hurdle model for determining the ability of an anabolic therapy toovercome the bone loss associated with hormone ablation (Zhou, S. et.al., Journal of Cellular Biochemistry, PMID: 11455579). Treatment withrecombinant R2M3-26 treatment reversed bone loss in anovariectomy-induced osteoporosis mouse model, as shown in FIGS. 27A-27C.Increased cortical thickness was observed in the trabecular regions,suggesting increased compressive strength. R2M3-26 treatment increasedfemur mid-diaphysis cortical bone thickness after 42 days as measured bymicro CT, as shown by FIG. 27D. BMD was also increased by R2M3-26 asmeasured by DEXA, shown in FIG. 27E.

A single injection of R2M3-26 was sufficient to induce rapid boneformation and bone volume within one week, as shown in FIGS. 28A-28C,wherein * indicates P value <0.05.

High dose treatment with R2M3-26 and 1R-C07-26 rapidly and significantlyincreased bone volume and bone mineral density, and improvedbiomechanical strength of the bone (ultimate load to failure andstiffness), as shown in FIGS. 29A-29D. 1R-C07-26 showed a robust andsignificant effect on bone accrual that persisted through 28 days. BothR2M3-26 and 1R-C07-26 significantly increased the resistance to fractureafter 28 days of treatment by biomechanical testing.

High dose treatment with R2M3-26 and 1R-C07-3 rapidly and significantlyincreased bone volume, bone mineral density, and cortical thicknessafter only 14 days of treatment, as shown in FIG. 30A-E. 1R-C07-3 at 10mpk appeared more effective at increasing bone mass than any othertreatment tested in this preclinical model.

These studies demonstrate that recombinant protein treatment can inducerapid and sustained increase of bone mineral density and bone volume innaive mice and mouse osteoporosis model. Both bone volume and bonemineral density (BMD) increased rapidly, suggesting resistance tofracture. IgG2-anti-GFP is a negative control. Anti-Beta Galactosidase(anti-βgal) was a negative control.

An additional experiment was done to determine systemic skeletal effectsof Wnt surrogate molecules in an ovariectomy-induced model ofosteoporosis. 057BL/6 females, 4 weeks old at time of ovariectomy,(n=8/group) were compared to sham surgery operated as well asage-matched naïve mice. Animals were injected i.p. with recombinant Wntsurrogate molecules 7 months after surgery and when the onset ofosteoporosis was confirmed. Experimental groups included R2M3-26,1RC07-3, anti-Bgal (Ab control), and vehicle (PBS). Sub-cutaneousinjection of romosozumab was done to another cohort of mice forcomparison. Animal were treated twice weekly, and followed for 4 weeks.

As shown in FIG. 31, whole body bone mineral density (BMD) was measuredweekly using dual-energy X-Ray absorptiometry (DEXA) and treatment withWnt surrogate molecules can not only reverse but even surpass the totalBMD seen in naïve or non-surgical animals. After 4 weeks of treatment,animals were assessed for the vertebral resistance to compressionfracture.

Treatment with Wnt surrogate molecules significantly increased thevertebral resistance to compression fracture, as shown by fractureanalysis (FIG. 32). 1RC07-3 most robustly increases the Max Forcerequired to compressively fracture the vertebra.

The Einhorn fracture model (Bonnarens F, Einhorn TA. J Orthop Res. 1984;2(1):97-101.PMID: 6491805) with delayed treatment with Wnt surrogatemolecules was used to determine ability of this therapy to inducefracture healing. Delayed treatment with either 1RCO7-3 or R2M3-26 wastested to determine if either molecule was capable of contributing toincreased fracture healing after a mid-traverse femoral fracture.C57BL/6 females, 16 weeks old at time of fracture (n=8/group) were used.The presence of a cartilaginous callus 2 weeks after fracture wasconfirmed in all animals before the start of treatment. With delayedtreatment to allow for callus formation, a pure osteogenic signal couldbe elicited, with rapid mineralization of the already existing callus.

Animals were injected i.p. with recombinant Wnt surrogate molecules withthe following experimental groups: R2M3-26, 1RCO7-3, anti-Bgal (Abcontrol), and vehicle (PBS). Sub-cutaneous injection of romosozumab wasdone to another cohort of mice for comparison. Animal were treated twiceweekly, and followed for 6 weeks. Radiography was used to visualizechanges in mineralization of the callus throughout the experiment (FIGS.33A and B). An increase in the mineralization and size of the resultingcallus with Wnt surrogate treatment is apparent at both 1 and 6 weeks oftreatment. One week was sufficient to induce rapid mineralization thatis predictive of rapid bone fracture healing and resistance to fracture.1RCO7-3 appeared to induce mineralization to a greater extent thanR2M3-26. Radiographs taken after 6 weeks of treatment show thepersistence of the highly mineralized callus in the 1RCO7-3 group, whilesome of the fracture callus in the R2M3-26 group has diminished (FIGS.33B and D).

Whole body DEXA was measured throughout the experiment to examine bonemineral density in not only the fractured femur, but also thecontralateral, non-fractured, femur where the expected increases in bonemineral density occurred after treatment (FIGS. 33C and D). Thisprovides reduced risk of secondary fracture in the already fracturedlimb and the appendicular skeleton. BMD of contralateral femur at 42days are shown in FIG. 33C.

Following 6-weeks of treatment after the confirmation of callus, wescanned the femurs with micro-computed tomography and determinedmultiple parameters which have been associated with an increasedresistance to fracture after healing. Callus tissue volume, the bonevolume within that callus, and especially bone mineral content are allsignificantly increased within the region of interest examined (FIG.33D). Qualitatively, the reconstructions show the prevalence of thickosteoid and mineral within the treated fracture. These parameterssuggest a robust resistance to fracture and indicate that delayedtreatment with Wnt surrogate molecules, after the spontaneouscartilaginous callus formation, can initiate a rapid and significantincrease in bone formation.

In another experiment, the dosing schedule was tested to determinewhether Wnt surrogate molecule therapy can induce a significant boneanabolic effect, how long that effect persists, and after washout, todetermine how the bone responds to additional treatment. Systemicskeletal effects were compared with variable dosing of 1RCO7-3 onanabolic effect, washout, and redosing after establishing baseline.C57BL/6 females, 12 weeks old (n=8/group) were injected i.p. withrecombinant Wnt surrogate molecules with the following experimentalgroups: two groups with 1 RCO7-3, one with anti-βgal (Ab control), andone with vehicle (PBS) on day 0 of the experiment. Sub-cutaneousinjection of romosozumab was done to another cohort of mice on day 0 forcomparison (romosozumab is an anti-sclerostin antibody (Saag et al., NEngl J Med. 2017 Oct. 12; 377(15):1417-1427; PMID:28892457) that mayreverse the bone loss associated with osteoporosis). Animals in 1 RC07-3treated groups had significant and rapid induction of bone formation by14 days (FIG. 34). One group received a second injection at day 14 todetermine if the bone anabolic effect could be further enhanced.Interestingly, regardless of treatment, all treatment effects werereversed and normalized after 35 days. A 2-week period was allowed toreturn to baseline levels prior to a second round of treatment. On day49, a second round of treatment was done to all experimental groups. TheWnt surrogate treated animals responded rapidly, however not to the samemagnitude as with the initial treatment (FIG. 34). For all groups, 5weeks after the last injection, new bone formation ceased. Thisindicates that a single injection is capable of significantly increasingbone formation, however the anabolic effect is rapidly lost. Thissuggests that an anti-resorptive agent may be required in combinationwith Wnt surrogate therapy to maintain the anabolic effect.

The mechanism of action of romosozumab relies upon the stimulation ofbone formation by removing an inhibitor (sclerostin) of endogenous Wntsignaling. An experiment was done treating animals with Wnt surrogatemolecules plus romosozumab to determine if Wnt surrogate moleculetreatment was capable of synergizing with romosozumab in a combinationstudy.

C57BL/6 males, 10 weeks old (n=8/group) were injected i.p. withrecombinant Wnt surrogate molecules in combination with romosozumab inthe following experimental groups: 1RC07-3 (0.1 mpk), 1RCO7-3 (1 mpk),1RCO7-3 (10 mpk), 1RCO7-3 (0.1 mpk)+romosozumab (25 mpk), 1RCO7-3 (1mpk)+romosozumab (25 mpk), 1RCO7-3 (10 mpk)+romosozumab (25 mpk),romosozumab (25 mpk) alone, anti-Bgal (Ab control), and vehicle (PBS).Animals were treated twice weekly and followed for 3 weeks.

Whole body BMD was measured weekly and results are presented in FIG. 35.Conclusions from this study are that endogenous romosozumab canstimulate additional bone growth in the presence of high dose 1 RC07-3.These data further suggest that peak anabolic action has not yet beenreached with 10 mpk 1RCO7-3 treatments. These data also show thatromosozumab can stimulate bone formation even in the presence of1RCO7-3. Overall, This study shows that Wnt surrogate molecule treatmentcan synergize with romosozmab to enhance the bone anabolic effect afteronly 21 days of twice weekly treatment.

Changes in gene expression in whole bone were measured in a time courseafter Wnt surrogate molecule therapy in mice to assess how this therapyregulates expression of genetic markers related to proliferation andosteogenesis. C57BL/6 females, 13 weeks old (n=5/group) were injectedi.p. with 1RCO7-3 or anti-Bgal (Ab control) once. Sub-cutaneousinjection of romosozumab was done to another cohort of mice forcomparison. Cohorts of animals were sacrificed at 8, 24, 48 and 120hours after treatment, and tibia bones and serum were isolated and flashfrozen for RNA extraction. ELISA was used to measure levels oftherapeutic molecules in serum over the course of the experiment asdescribed above (FIG. 36).

For purification of RNA from bone, the excised tibia from freshlysacrificed animals were processed as follows: the ends of the tibia wereclipped to expose marrow cavity and marrow cavity was flushed with icecold saline through 30 gauge needle, Steps were taken to ensure allmuscle tissue and cartilage has been removed; bone appeared completelywhite with no red marrow component residual. Tibias were placed in 1.5mL Eppendorf tubes and flash frozen in liquid nitrogen. To lyse, asingle tissue lyser bead was placed in the tube with bone and Trizol wasadded directly to frozen bone and bead. Tissue lyser at high speed wasused to completely homogenize. Homogenates were then subject tochloroform extraction to separate the nucleic acid phase. Furtherisolation and purification was carried out using an RNeasy mini kit(Qiagen).

RNA Isolated from tibias was tested for relative transcript levels ofRunt Related Transcription Factor 2 (RunX2), Collagen Type I Alpha 1Chain (Col1A1), Dentin Matrix Acidic Phosphoprotein 1 (Dmp1), Alkalinephosphatase (Alp), Receptor activator of nuclear factor kappa-B ligand(RankL), Dickkopf WNT Signaling Pathway Inhibitor 1 (Dkk1), sclerostin(Sost), Cyclin D1 (Ccnd1), Axin2, and Ki67.

TABLE 4 Changes in gene expression in bone with Wnt surrogate molecule(1RC07-3) and romosozumab (Rxmab) Therapy in mice Fold Induction + /−SEM relative to Bgal mean value gene Time(hr) Bgal Rzmab 1RC073 RunX2 241.0 + /− 0.3  8.8 + /− 4.4   9.6 + /− 3.6**   48 1.0 + /− 0.7  1.9 + /−1.0   1.5 + /− 0.5     ColA1 24 1.0 + /− 0.2  3.9 + /− 2.0   3.0 + /−1.0     48 1.0 + /− 0.5  7.0 + /− 3.3   2.7 + /− 1.5     Dmp1 24 1.0 +/− 0.2  2.7 + /− 1.1   4.8 + /− 2.9     48 1.0 + /− 0.6  1.6 + /− 0.7  3.8 + /− 1.8     Alp 24 1.0 + /− 0.4 18.3 + /− 11.7 16.5 + /− 6.4*   48 1.0 + /− 0.6  4.0 + /− 1.8   5.0 + /− 1.9     RankL 24 1.0 + /− 0.3 8.6 + /− 4.5   8.7 + /− 3.3     48 1.0 + /− 0.7  1.5 + /− 0.6   4.3 +/− 1.4     Dkk1 24 1.0 + /− 0.2 10.5 + /− 5.1  17.9 + /− 6.0**   481.0 + /− 0.5  4.3 + /− 2.5   2.8 + /− 1.3     Sost 24 1.0 + /− 0.2 2.1 + /− 1.0   2.5 + /− 0.9     48 1.0 + /− 0.6  5.1 + /− 2.3   9.0 +/− 4.2**   Ccnd1 24 1.0 + /− 0.4  3.3 + /− 0.8   3.4 + /− 1.8     481.0 + /− 0.6  2.5 + /− 1.2   2.4 + /− 1.2     Axin2 24 1.0 + /− 0.5 3.2 + /− 1.1   6.1 + /− 1.8**   48 1.0 + /− 0.8  3.6 + /− 2.3   4.6 +/− 1.6*    Ki67 24 1.0 + /− 0.3  7.7 + /− 4.3  10.0 + /− 3.2     481.0 + /− 0.5  1.0 + /− 0.5   25.5 + /− 12.2 *** * p<0.05, ** p<0.005,2-way ANOVA comparison with Bgal control *** p<0.001, 2-way ANOVAcomparison with both Bgal and romosozumab

In comparison to anti-sclerostin antibody (romosozumab) treatment, geneexpression signatures over the time points were distinct with Wntsurrogate molecule therapy, with induction of more robust Axin2 and Ki67expression than that caused by romosozumab treatment.

Example 22 In Vivo Liver Regeneration Model and Characterization ofAAV-Delivered Wnt Surrogates

In vivo experiments were conducted by infecting approximately 8-weeksold C57BL/6J mice with an AAV vector that expressed Flag- and His-tagged18R5-DKK1c protein (AAV-18R5-DKK1c-FlagHis). 18R5-DKK1c is a fusionprotein containing the frizzled binding antibody, 18R5, in scFv formatfused to DKK1c, as described in PCT Publication WO2016/040895, e.g.,FIG. 5. Control mice were either injected sub-cutaneously withphosphate-buffered saline (PBS) only or romosozumab (10 mg/kg), orinjected intravenously (IV) with an AAV vector that expressed greenfluorescent protein (GFP) (AAV-CAG-GFP), or an AAV vector that expresseda fusion protein comprising an anti-GFP scFv fused to a mutant DKK1c(AAV-ScFv (anti-GFP)-DKK1cF234K-Flag-His). 28 days after infection,animals were weighed and sacrificed. The liver was weighed, and theliver to body weight ratio calculated. The content of the smallintestine and colon was removed by flushing with phosphate-bufferedsaline and gentle pressure to expel the content. The small intestine andcolon were then weighed.

Systemic expression of 18R5-DKK1c-FlagHis resulted in a significantliver weight increase (FIG. 37A). Systemic expression of the negativecontrols, eGFP or anti-eGFP-Dkk1cF234K, did not affect the liver to bodyweight ratio. Administration of the romosozumab recombinant protein orvehicle control, did not affect the liver to body weight ratio.

None of the treatments affected the small intestine (FIG. 37B) or colon(FIG. 37C) to body weight ratio.

These studies showed that 18R5-DKK1c-FlagHis increases liver weight, butnot that of the small or large intestines. This suggests that18R5-DKK1c-FlagHis can promote liver regeneration.

Example 23 In Vivo Liver Regeneration Model and Characterization ofRecombinantly Produced Wnt Surrogates

In vivo experiments were conducted by treating mice with recombinantlyproduced anti-eGFP, R2M3-26, 1R-C07-26, romosozumab or Rspo2 proteins atvarious dosages. The Rspo2 protein is a fusion protein between a shortsplice variant of the Rspo2 gene and a human Fc fragment.

In one study, mice were housed 4 per cage and n=8 per treatment groupwere used. Approximately 8-weeks old C57BL6/J mice were administeredrecombinant proteins, anti-eGFP (1 mg/kg), R2M3-26 (1 or 10 mg/kg) or1R-C07-26 (1, 5 or 10 mg/kg), twice weekly intra-peritoneally (i.p.) forfour weeks. In addition, groups of mice were administered romosozumab(30 mg/kg) or PBS vehicle control, subcutaneously.

Mice were weighed at the beginning and throughout the treatment. None ofthe treatment with recombinant proteins affected total body weightsignificantly (FIG. 38A). On day 28, the liver was weighed, and theliver to body weight ratio calculated (FIG. 38B). The highest dose ofR2M3-26 (10 mg/kg) resulted in a significant increase in liver to bodyweight ratio. None of the other treatment affected the liver weightsignificantly.

The increase in liver weight in response to R2M3-26 suggest that thisrecombinant protein can promote liver regeneration.

In another study, mice were housed 5 per cage and n=10 per treatmentgroup were used. Approximately 8-weeks old C57BL/6J mice received asingle i.p. injection containing anti-eGFP (0.56 mg/kg), R2M3-26 (0.3mg/kg) or Rspo2 (0.46 mg/kg) alone, or with a combination of R2M3-26(0.1 mg/kg) and Rspo2 (0.46 mg/kg).

24 or 48 hours after injection, mice were euthanized. A portion of theleft liver lobe was snap-frozen in liquid nitrogen and stored at −80° C.for RNA analysis. Cyclin D1 and Ki67 expression were measured byperforming qPCR using the Mm00432359_m1 Ccnd1 probe and theMm01278617_m1 Ki67 probe (Thermofisher, 4331182). An additional portionof the left liver lobe was fixed in formalin and embedded in paraffinfor immunohistochemistry analysis. Sections were stained with theanti-proliferating cell nuclear antigen (PCNA) (Abcam, ab18197) oranti-phospho-histone H3 (pH3) rabbit antibodies (Abcam, ab47297). Thenumber of positive nuclei were counted using the image processingsoftware, Image J.

Rspo2 alone increased Ki67 (FIG. 39A) and CyclinD1 (FIG. 39B) mRNAexpression. In combination with R2M3-26, Rspo2 increased Ki67 andCyclinD1 expression further than with Rspo2 alone at 24 and 48 hoursafter treatment with recombinant proteins. Rspo2 alone increased thenumber of PCNA (FIG. 39C) and pH3 (FIG. 39D) positive nuclei in liversections. In combination with R2M3-26, Rspo2 increased the number ofPCNA and pH3 positive nuclei further than with Rspo2 alone at 48 hoursafter treatment with recombinant proteins.

These studies show that the proliferation markers, Ki67 mRNA, CyclinD1mRNA and PCNA-positive nuclei, and the pH3 mitotic marker are induced bythe R2M3-26 and Rspo2 recombinant proteins and suggest that theserecombinant proteins can promote liver regeneration.

Example 24 In Vivo Chronic Liver Injury Model and Characterization ofAAV-Delivered Wnt Surrogates

Two in vivo experiments were conducted in two, thioacetamide (TAA)- andCC14-induced, liver cirrhosis mouse models, thioacetamide (TAA)- andCC14-induced, to test the effect of AAV vectors expressing18R5-DKK1c-FlagHis or Rspo2 protein on chronic liver injury. TAA wasadded to the drinking water of 6 weeks old C57BL/6J mice at aconcentration of 300 mg/L throughout the TAA treatment duration. Micewere housed 5 per cage and groups of n=10 were used, except for thecontrol groups without TAA treatment where n=5 per group was used.

In Study 1 (FIGS. 40A, 40C, 40E, 40G-H), mice with (n=10) or without(n=5) TAA treatment were weighed and sacrificed after 9 weeks of TAAaddition into the drinking water, to measure baseline values. Liverswere weighed and liver samples were collected for mRNA and histologicalanalysis. TAA supplementation was maintained in the drinking water ofthe remaining mice and they were injected IV with AAV vectors thatexpressed an enhanced green fluorescent protein (eGFP) (3e10 genomicparticles (GC)), 18R5-DKK1c-FlagHis (3e10 or 1e11 GC) or Rspo2 protein(1e11 GC) or a combination of 18R5-DKK1c-FlagHis (3e10 GC) and Rspo2(1e11 GC). 5 age-matched naïve animals (no TAA) were kept as a negativecontrol. Three weeks after AAV injection, all mice were weighed andeuthanized. Livers were weighed and liver samples were collected formRNA and histological analysis.

Treatment with 18R5Dkk1FH or Rspo2 resulted in a significant increase ofliver weight (FIG. 40C) and liver to body weight ratio (FIG. 40E) inmice undergoing a continuous exposure to TAA. Treatment with acombination of 18R5Dkk1FH and Rspo2 resulted in a further increase inliver weight and liver to body weight ratio beyond that observed witheither treatment alone. The combination 18R5Dkk1FH and Rspo2 treatmentresulted in a decrease of the fibrosis marker Col1a1 mRNA expression(FIG. 40G). Histological liver sections were stained with Sirius red tovisualize the accumulation of collagen in fibrotic area (FIG. 40H).Quantification of the percentage of red are, using Image J analysissoftware, showed a significant increase of fibrotic area in TAA-treatedmice. A combination of 18R5Dkk1FH and Rspo2 resulted in a reversal offibrotic area increase when compared to mice treated with the eGFPnegative control. Treatment with Rspo2 alone also resulted in asignificant but smaller reversal than the combination treatment.

In Study 2, (FIGS. 40B, 40D, 40F), mice were exposed to TAA-supplementedwater for 11 weeks, and were returned to standard drinking thereafter,two days prior to AAV treatment. At the start of AAV treatment, miceexposed to TAA (n=10) or not (n=5) were weighed and sacrificed tocollect liver samples for baseline measurements. The remaining mice wereinjected with AAV vectors that expressed an enhanced green fluorescentprotein (eGFP) (1.3e11 genomic particles (GC)), 18R5-DKK1c-FlagHis (3e10or 1e11 GC) or Rspo2 protein (1e11 GC) or a combination of18R5-DKK1c-FlagHis (3e10 GC) and Rspo2 (1e11 GC). 5 age-matched naïveanimals (no TAA) were kept as a negative control. Three weeks after AAVinjection, all mice were weighed and euthanized. Livers were weighed andliver samples were collected for mRNA and histological analysis.

A similar liver weight and liver to body weight increase was observed inmice treated with 18R5-Dkk1c-FlagHis and Rspo2 either alone or incombination than what was observed in Study 1 (FIGS. 40D, 40F).

These studies show that 18R5-Dkk1c-FlagHis and Rspo2 can increase liverweight in a TAA-induced liver cirrhosis model and reduce fibrosismarkers. These results suggest that 18R5-Dkk1c-FlagHis and Rspo2 canpromote liver tissue repair after chronic liver injury.

Example 25 In Vivo Chronic Liver Injury Models and Characterization ofRecombinantly Produced Wnt Surrogates

In vivo experiments were conducted in thioacetamide (TAA)-induced andCC14-induced mouse models of liver cirrhosis by treating mice withrecombinantly produced anti-eGFP, R2M3-26 and Rspo2 proteins.

In the TAA-induced cirrhosis model, six weeks old male mice were exposedto TAA-supplemented drinking water (300 mg/L) for approximately 22 weeks(FIG. 41A). TAA exposure was removed two days prior to beginning oftreatment with recombinant proteins and mice were provided with freshdrinking water. Mice were housed 5 per cage and n=10 per treatment groupwere used. In a mono treatment study (FIGS. 41B, 41D, 41F, 41H, 41J,41L), mice were injected i.p. with anti-eGFP (1 mg/kg) or Rspo2 (1mg/kg) twice weekly. In a combination treatment study (FIGS. 41C, 41E,41G, 41I, 41K, 41M), mice were injected i.p. with anti-eGFP (1.3 mg/kg)or a combination of R2M3-26 (0.3 mg/kg) and Rspo2 (1 mg/kg) twiceweekly. Mice were then weighed and sacrificed at day 3, 7 or 14 daysafter beginning of treatment. Groups of control mice without exposure toTAA (n=5 per group) were euthanized at day 0 and day 14 in both studies.

Treatment with Rspo2 protein alone or in combination with R2M3-26resulted in an increase in liver to body weight ratio (FIGS. 41B and41C) and a transient stimulation of the Wnt signaling pathway as shownby an increase in Axin2 expression (FIGS. 41D and 41E). Treatment withRspo2 protein alone or in combination with R2M3-26 induced the followingproliferation markers: cyclinD1 (FIGS. 41F, 41G) and Ki67 (FIGS. 41H and41I) mRNA expression, PCNA (FIGS. 41J, 41K) and pH3 (FIGS. 41L, 41M)positive nuclei.

In an additional study, plasma was collected for prothrombin timemeasurement. Coagulation time is impaired in TAA-induced cirrhosis modelas exemplified by an increase in pro-thrombin (PT) test to normal valueratio in mice exposed to TAA when compared to normal mice without TAAexposure (FIG. 41N). Treatment with Rspo2 (1 mg/kg) and R2M3-26 (0.3mg/kg) reversed the prolongation in PT time as shown by the decrease inPT_(test)/PT_(normal) ratio at day 7 and day 14 after biweekly Rspo2 andR2M3-26 treatment.

These studies show that Rspo2 and R2M3-26 can stimulate liver cellproliferation and improve hepatocytes functional activity such aspro-thrombin time in a TAA-induced liver cirrhosis model. These resultssuggest that Rspo2 and R2M3-26 can promote liver tissue repair inchronic liver disease.

In the CC14-induced cirrhosis model, six-weeks old C57BL/6J male micewere injected i.p. with 2 ml/kg CC14 in mineral oil, twice weekly for 8weeks (FIG. 42A). 3 days after the last CC14 injection, mice wereinjected i.p. twice weekly with the following recombinant proteins:anti-β-galactosidase (10 mg/kg), Rspo2 (1 or 10 mg/kg), or a combinationof R2M3-26 (0.3 mg/kg) and Rspo2 (1 mg/kg). Three additional controlgroups were included: one group injected with CC14 but no proteins, onegroup injected with mineral oil, and one untreated age-matched naïvegroup. n=8 were used for each group. After two weeks of treatment withrecombinant proteins, mice were weighed and sacrificed. Plasma wascollected for pro-thrombin time measurement. Livers were weighed andliver samples were collected for histological analysis.

Treatment with R2M3-26 and Rspo2 resulted in a significant increase inliver to body weight ratio when compared to treatment withanti-β-galactosidase negative control (FIG. 42B). Treatment with Rspo2(10 mg/kg) or a combination of R2M3-26 and Rspo2 resulted in asignificant decrease in prothrombin time (FIG. 42C). Treatment withRspo2 (10 mg/kg) or a combination of R2M3-26 and Rspo2 resulted in asignificant reversal in fibrotic area, induced by CC14 (FIG. 42D).

This study showed that Rspo2 and R2M3-26 can induce an increase in liverweight, improve hepatocytes functional activity such as pro-thrombintime and reduce fibrosis markers in a CC14-induced liver cirrhosismodel. These results suggest that Rspo2 and R2M3-26 can promote livertissue repair in chronic liver disease.

Example 26 In Vivo Acute Liver Injury Model and Characterization ofRecombinantly Produced Wnt Surrogates

In vivo experiments were conducted using acetaminophen-induced mousemodels of acute liver injury by treating mice with recombinantlyproduced anti-eGFP, R2M3-26 and Rspo2 proteins.

Eight-week old C57BL/6 male mice were housed 5 per cage. n=10 were usedper group. Mice were fasted overnight for 12 hours. Acetaminophen (APAP)was administered i.p. at a sublethal dose (300 mg/kg).

In a first study, anti-eGFP (0.3 mpk) or R2M3-26 (0.3 mpk) were injectedi.p. either immediately after or 3 or 6 hours after APAP injection (FIG.43). Serum samples were collected at 24 and 48 hours after APAPinjection for ALT measurements. Mice were sacrificed 48 hours after APAPinjection and liver samples were collected for mRNA analysis.

R2M3-26 treatment did not affect ALT level significantly (FIG. 43B).Treatment with R2M3-26 induced cyclinD1 (FIG. 43C) and Ki67 (FIG. 43D)mRNA significantly, beyond the level induced by APAP treatment alone.

In a second study, human Fc (0.46 mg/kg) or Rspo2 (0.46 mg/kg) wereinjected i.p. either immediately after or 3 or 6 hours after APAPinjection (FIG. 44). Serum samples were collected at 24 and 48 hoursafter APAP injection. Liver samples were collected at 48 hours afterAPAP injection. Rspo2 treatment did not affect ALT serum levelsignificantly (FIG. 44B). Treatment with Rspo2 induced cyclinD1 (FIG.44C) and Ki67 (FIG. 43D) mRNA significantly, beyond the level induced byAPAP treatment alone.

In a third study, anti-eGFP (0.56 mg/kg) or a combination of R2M3-26(0.1 mg/kg) and Rspo2 (0.46 mg/kg) were injected i.p. 3 hours after APAPadministration. Serum and liver samples were collected at 24, 36, 48 and60 hours after APAP injection for ALT measurements and mRNA analysis.

R2M3-26 and Rspo2 combination treatment did not affect ALT levelsignificantly (FIG. 45B). Treatment with R2M3-26 and Rspo2 inducedcyclinD1 (FIG. 45C) and Ki67 (FIG. 45D) mRNA significantly, beyond thelevel induced by APAP treatment alone.

An additional study was performed to evaluate the effect of Rspo2 andR2M3-26 on the survival of mice treated with a dose of 600 mg/kg ofacetaminophen (FIG. 46). Anti-eGFP (0.3 mg/kg), R2M3-26 (0.3 mg/kg),Rspo2 (0.46 mg/kg) or a combination of R2M3-26 (0.1 mg/kg) and Rspo2(0.46 mg/kg) were injected i.p. 3 hours after APAP administration. Micewere monitored several times daily over the next 96 hours. A consistenttrend in improving survival was observed in groups treated with R2M3-26(FIG. 46B), Rspo2 (FIG. 46C) or a combination of R2M3-26 and Rspo2 (FIG.46D).

These studies show that Rspo2 and R2M3-26 can induce proliferationmarkers beyond that induced spontaneously in APAP-induced acute injurymodel. These results suggest that Rspo2 and R2M3-26 can enhance livertissue repair after an acute liver injury.

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments. These and other changes can be made to the embodiments inlight of the above-detailed description.

In general, in the following claims, the terms used should not beconstrued to limit the claims to the specific embodiments disclosed inthe specification and the claims, but should be construed to include allpossible embodiments along with the full scope of equivalents to whichsuch claims are entitled. Accordingly, the claims are not limited by thedisclosure.

What is claimed is:
 1. A soluble, multivalent, multispecific Wntsurrogate molecule, wherein the Wnt surrogate molecule comprises: (i)one or more region that specifically binds to one or more Frizzled (Fzd)receptor (a Fzd binding region); and (ii) one or more region thatspecifically binds to a Low-density lipoprotein (LDL) receptor-relatedprotein 5 (LRP5) and/or a Low-density lipoprotein (LDL) receptor-relatedprotein 6 (LRP6) (a LRP5/6 binding region).
 2. The Wnt surrogatemolecule of claim 1, comprising one or more Fzd binding regions and oneor more LRP5/6 binding regions.
 3. The Wnt surrogate molecule of claim1, wherein the one or more Fzd binding regions comprise one or moreantigen-binding fragments of an antibody.
 4. The Wnt surrogate moleculeof claim 3, wherein the one or more antigen-binding fragments areselected from the group consisting of: IgG, scFv, Fab, and VHH or singledomain antibodies (sdAb).
 5. The Wnt surrogate molecule of any of claims3-4, wherein the one or more Fzd antigen-binding fragments comprise: (i)CDRH1, CDRH2 and CDRH3 sequences set forth for any of the antibodies ofTables 1A or 1B; and/or (ii) CDRL1, CDRL2 and CDRL3 sequences set forthfor any of the antibodies of Tables 1A or 1B, or a variant of said Fzdbinding region comprising one or more amino acid modifications, whereinsaid variant comprises less than 8 amino acid substitutions in said CDRsequences.
 6. The Wnt surrogate molecule of any of claims 3-5, whereinthe one or more Fzd binding regions comprise an amino acid sequencehaving at least 90% identity to any of the sequences set forth in SEQ IDNOs:1-65 or 129-132, or an antigen-binding fragment thereof.
 7. The Wntsurrogate molecule of any of claims 1-6, wherein the one or more Fzdbinding regions bind to one or more of Frizzled 1 (Fzd1), Frizzled 2(Fzd2), Frizzled 3 (Fzd3), Frizzled 4 (Fzd4), Frizzled 5 (Fzd5),Frizzled 6 (Fzd6), Frizzled 7 (Fzd7), Frizzle 8 (Fzd8), Frizzled 9(Fzd9), and Frizzled 10 (Fzd10).
 8. The Wnt surrogate molecule of claim7, wherein the one or more Fzd binding region binds to two or more ofFrizzled 1 (Fzd1), Frizzled 2 (Fzd2), Frizzled 3 (Fzd3), Frizzled 4(Fzd4), Frizzled 5 (Fzd5), Frizzled 6 (Fzd6), Frizzled 7 (Fzd7),Frizzled 8 (Fzd8), Frizzled 9 (Fzd9), and Frizzled 10 (Fzd10).
 9. TheWnt surrogate molecule of claim 8, wherein the one or more Fzd bindingregion binds to: (i) Fzd1, Fzd2, Fzd7 and Fzd9; (ii) Fzd1, Fzd2 andFzd7; (iii) Fzd5 and Fzd8; (iv) Fzd5, Fzd7 and Fzd8; (v) Fzd1, Fzd4,Fzd5 and Fzd8; (vi) Fzd1, Fzd2, Fzd5, Fzd7 and Fzd8; (vii) Fzd4 andFzd9; (viii) Fzd9 and Fzd10; (ix) Fzd5, Fzd8 and Fzd10; or (x) Fzd4,Fzd5 and Fzd8; Fzd1, Fzd5, Fzd7 and Fzd8.
 10. The Wnt surrogate moleculeof any of claims 1-9, wherein the one or more LRP5/6 binding regionscomprise one or more antigen-binding fragments of an antibody.
 11. TheWnt surrogate molecule of claim 10, wherein the one or moreantigen-binding fragments are selected from the group consisting of:IgG, scFv, Fab, and VHH or sdAb.
 12. The Wnt surrogate molecule of anyof claims 1-11, wherein the one or more LRP5/6 binding regions orantigen-binding fragments comprise: (i) CDRH1, CDRH2 and CDRH3 sequencesset forth for any of the antibodies of Table 2; and/or (ii) CDRL1, CDRL2and CDRL3 sequences set forth for any of the antibodies of Table 2, or avariant of said LRP5/6 binding region comprising one or more amino acidmodifications, wherein said variant comprises less than 8 amino acidsubstitutions in said CDR sequences.
 13. The Wnt surrogate molecule ofany of claims 10-12, wherein the one or more LRP5/6 binding regionscomprise an amino acid sequence having at least 90% identity to any ofthe sequences set forth in SEQ ID NOs:66-88 or 133, or anantigen-binding fragment thereof.
 14. The Wnt surrogate of any of claims1-13, wherein the Fzd binding region and the LRP5/6 binding regioncomprise the sequences set forth in Table 3 for any of the Wntsurrogates disclosed therein.
 15. The Wnt surrogate molecule of any ofclaims 1-14, wherein the Fzd binding region comprises a Fab, and theLRP5/6 binding region comprises a VHH or sdAb or a scFv.
 16. The Wntsurrogate molecule of claim 15, wherein the Fab is present within a fullimmunoglobulin (Ig), optionally an IgG, comprising a light chain and aheavy chain.
 17. The Wnt surrogate molecule of claim 16, wherein theLRP5/6 binding region is fused to the N-terminus or the C-terminus ofthe heavy chain or is fused to the N-terminus or the C-terminus of thelight chain.
 18. The Wnt surrogate molecule of claim 17, wherein theLRP5/6 binding region is fused to the heavy chain or the light chain viaone or more linker moiety.
 19. The Wnt surrogate molecule of any ofclaims 1-14, wherein the Fzd binding region comprises a VHH or sdAb orscFv, and the LRP5/6 binding region comprises a Fab.
 20. The Wntsurrogate molecule of claim 19, wherein the Fab is present within a fullimmunoglobulin (Ig), optionally an IgG, comprising a light chain and aheavy chain.
 21. The Wnt surrogate molecule of claim 20, wherein the Fzdbinding region is fused to the N-terminus or the C-terminus of the heavychain.
 22. The Wnt surrogate molecule of claim 20, wherein the Fzdbinding region is fused to the N-terminus or the C-terminus of the lightchain.
 23. The Wnt surrogate molecule of claim 21 or claim 22, whereinthe Fzd binding region is fused to the heavy chain or the light chainvia one or more linker moiety.
 24. The Wnt surrogate molecule of any ofclaims 1-14, wherein the Fzd binding region comprises a Fab or Fv, andthe LRP5/6 binding region comprises a Fab or Fv.
 25. The Wnt surrogatemolecule of claim 24, wherein the Fab of the Fzd binding region or theFab of the LRP5/6 binding region is present within a full immunoglobulin(Ig), optionally an IgG, comprising a light chain and a heavy chain. 26.The Wnt surrogate molecule of claim 25, wherein only one of the Fab ofthe Fzd binding region or the Fab of the LRPp5/6 binding region ispresent within the full immunoglobulin (Ig).
 27. The Wnt surrogatemolecule of claim 26, wherein the Fab of the Fzd binding region ispresent within the full Ig.
 28. The Wnt surrogate molecule of claim 27,wherein the Fab or Fv of the LRP5/6 binding region is fused to theN-terminus of the Ig, optionally the N-terminus of the heavy chain ofthe full Ig or the N-terminus of the light chain of the full Ig.
 29. TheWnt surrogate molecule of claim 27, wherein the Fab of the LRP5/6binding region is fused to the C-terminus of the Ig, optionally theC-terminus of the heavy chain of the full Ig or the C-terminus of thelight chain of the full Ig.
 30. The Wnt surrogate molecule of claim 27,wherein the variable light chain region of the LRP5/6 binding Fab isfused to the N-terminus of the variable heavy chain region of the fullIg.
 31. The Wnt surrogate molecule of claim 27, wherein the variablelight chain region of the LRP5/6 binding Fab is fused to the N-terminusof the variable heavy chain region of the full Ig, and the variableheavy chain region of the LRP5/6 binding Fab is fused to the N-terminusof the variable light chain region of the full IgG.
 32. The Wntsurrogate molecule of claim 27, wherein the variable light chain regionof the LRP5/6 binding Fv is fused to the N-terminus of the variableheavy chain region of the full Ig, and the variable heavy chain regionof the LRP5/6 binding Fv is fused to the N-terminus of the variablelight chain region of the full IgG.
 33. The Wnt surrogate molecule ofclaim 27, wherein the variable heavy chain region of the LRP5/6 bindingFv is fused to the N-terminus of the variable heavy chain region of thefull Ig, and the variable light chain region of the LRP5/6 binding Fv isfused to the N-terminus of the variable light chain region of the fullIgG.
 34. The Wnt surrogate molecule of claim 26, wherein the Fab of theLRP5/6 binding region is present within the full Ig.
 35. The Wntsurrogate molecule of claim 34, wherein the Fab or Fv of the Fzd bindingregion is fused to the N-terminus of the Ig, optionally the N-terminusof the heavy chain of the full Ig or the N-terminus of the light chainof the full Ig.
 36. The Wnt surrogate molecule of claim 34, wherein theFab or Fv of the Fzd binding region is fused to the C-terminus of theIg, optionally the C-terminus of the heavy chain of the full Ig or theC-terminus of the light chain of the full Ig.
 37. The Wnt surrogatemolecule of claim 34, wherein the variable light chain region of the Fzdbinding Fab is fused to the N-terminus of the variable heavy chainregion of the full Ig.
 38. The Wnt surrogate molecule of claim 34,wherein the variable light chain region of the Fzd binding Fab is fusedto the N-terminus of the variable heavy chain region of the full Ig, andthe variable heavy chain region of the Fzd binding Fab is fused to theN-terminus of the variable light chain region of the full IgG.
 39. TheWnt surrogate molecule of claim 34 or claim 35, wherein the variablelight chain region of the Fzd binding Fv is fused to the N-terminus ofthe variable heavy chain region of the full Ig, and the variable heavychain region of the Fzd binding Fv is fused to the N-terminus of thevariable light chain region of the full IgG.
 40. The Wnt surrogatemolecule of claim 34 or claim 35, wherein the variable heavy chainregion of the Fzd binding Fv is fused to the N-terminus of the variableheavy chain region of the full Ig, and the variable light chain regionof the Fzd binding Fv is fused to the N-terminus of the variable lightchain region of the full IgG.
 41. The Wnt surrogate molecule of any ofclaims 1-14, wherein the Fzd binding region comprises a VHH or sdAb orscFv, and the LRP5/6 binding region comprises a VHH or sdAb or scFv. 42.The Wnt surrogate molecule of claim 41, wherein the Fzd binding regionis fused to the Lrp5/6 binding region, and wherein the Fzd bindingregion or the LRP5/6 binding region is fused to an Fc region.
 43. TheWnt surrogate molecule of claim 41, wherein the Fzd binding region isfused to the N-terminus of an Fc region, and wherein the LRP5/6 bindingregion is fused to the C-terminus of an Fc region.
 44. The Wnt surrogatemolecule of claim 41, wherein the Fzd binding region is fused to theC-terminus of an Fc region, and wherein the LRP5/6 binding region isfused to the N-terminus of an Fc region.
 45. The Wnt surrogate moleculeof any of claims 1-14, wherein the surrogate molecule has a structureset forth in FIG. 1A-D, 10A, 15A, 16A or 18A.
 46. The Wnt surrogatemolecule of any of claims 1-45, wherein the one or more antigen-bindingfragment thereof, is humanized.
 47. The Wnt surrogate molecule of any ofclaims 1-46, which binds to the one or more Fzd receptor with a K_(D) of50 μM or lower.
 48. The Wnt surrogate molecule of any of claims 1-47,which binds to the one or more LRP5/6 receptor with a K_(D) of 50 μM orlower.
 49. The Wnt surrogate molecule of any of claims 1-48, whichmodulates a Wnt signaling pathway in a cell, optionally wherein the cellis a mammalian cell.
 50. The Wnt surrogate of claim 49, wherein the Wntsignaling pathway is a canonical Wnt signaling pathway or anon-canonical Wnt signaling pathway.
 51. The Wnt surrogate molecule ofclaim 49 or claim 50, which increases signaling via the Wnt signalingpathway in the cell.
 52. An isolated polynucleotide encoding apolypeptide sequence comprising one or more of the Fzd binding regionsand/or one or more of the LRP5/6 binding regions of a Wnt surrogatemolecule of any of claims 1-51.
 53. An expression vector comprising theisolated polynucleotide of claim
 52. 54. An isolated host cellcomprising the expression vector of claim
 53. 55. A pharmaceuticalcomposition comprising a physiologically acceptable excipient, diluent,or carrier, and a therapeutically effective amount of the Wnt surrogatemolecule according to any of claims 1-51, the polynucleotide of claim52, the expression cell of claim 53, or the host cell of claim
 54. 56. Amethod for agonizing a Wnt signaling pathway in a cell, comprisingcontacting the cell the Wnt surrogate molecule according to any ofclaims 1-51, the polynucleotide of claim 52, the expression cell ofclaim 53, or the host cell of claim 54, wherein the Wnt surrogatemolecule is an agonist of a Wnt signaling pathway.
 57. A method fortreating a subject having a disease or disorder associated with reducedWnt signaling, comprising administering to the subject an effectiveamount of the Wnt surrogate molecule according to any of claims 1-51,the polynucleotide of claim 52, the expression cell of claim 53, thehost cell of claim 54, or the pharmaceutical composition of claim 55,wherein the Wnt surrogate molecule is an agonist of a Wnt signalingpathway.
 58. The method of claim 57, wherein the disease or disorder isa bone disease or disorder.
 59. The method of claim 58, wherein the Wntsurrogate molecule binds Fzd1, Fzd2, and FZD7, and binds LRP5 and/orLRP6.
 60. The method of claim 59, wherein the Wnt surrogate moleculealso binds Fzd5 and Fzd8.
 61. The method of claim 57, wherein thedisease or disorder is selected from the group consisting of: bonefractures, stress fractures, vertebral compression fractures,osteoporosis, osteoporotic fractures, non-union fractures, delayed unionfractures, spinal fusion, pre-operative optimization for spinesurgeries, osteonecrosis, osseointegration of implants or orthopedicdevices, osteogenesis imperfecta, bone grafts, tendon repair,tendon-bone integration, tooth growth and regenerationmaxillofacialsurgery, dental implantation, periodontal diseases, maxillofacialreconstruction, osteonecrosis of the jaw, hip or femoral head, avascularnecrosis, alopecia, hearing loss, vestibular hypofunction, maculardegeneration, age-related macular degeneration (AMD), vitreoretinopathy,retinopathy, diabetic retinopathy, diseases of retinal degeneration,Fuchs' dystrophy, cornea diseases, stroke, traumatic brain injury,Alzheimer's disease, multiple sclerosis, muscular dystrophy, muscleatrophy caused by sarcopenia or chachexia, diseases affecting bloodbrain barrier (BBB) integrity, spinal cord injuries, spinal corddiseases, oral mucositis, short bowel syndrome, inflammatory boweldiseases (IBD), metabolic syndrome, diabetes, dyslipidemia pancreatitis,exocrine pancreatic insufficiency, wound healing, diabetic foot ulcers,pressure sores, venous leg ulcers, epidermolysis bullosa, dermalhypoplasia, myocardial infarction, coronary artery disease, heartfailure, hematopoietic cell disorders, immunodeficiencies, graft versushost diseases, acute kidney injuries, chronic kidney diseases, chronicobstructive pulmonary diseases (COPD), idiopathic pulmonary fibrosis,acute liver failure of all causes, acute liver failure drug-induced,alcoholic liver diseases including alcoholic hepatitis, chronic liverfailure of all causes, cirrhosis, liver fibrosis of all causes, portalhypertension, chronic liver insufficiency of all causes, nonalcoholicsteatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD) (fattyliver), alcoholic hepatitis, hepatitis C virus-induced liver diseases(HCV), hepatitis B virus-induced liver diseases (HBV), other viralhepatitis (e.g., hepatitis A virus-induced liver diseases (HAV) andhepatitis D virus-induced liver diseases (HDV)), primary biliarycirrhosis, autoimmune hepatitis, livery surgery, liver injury, livertransplantation, “small for size” syndrome in liver surgery andtransplantation, congenital liver disease and disorders, any other liverdisorder or detect resulting from genetic diseases, degeneration, aging,drugs, or injuries.
 62. A method for increasing bone mineral density,increasing bone volume, increasing bone cortical thickness, increasingbone mineral apposition rate, increasing bone stiffness, increasing bonebiomechanical strength, increasing resistance to bone fracture, ordecreasing bone loss associated with osteoporosis, comprising providingto a subject an effective amount of the pharmaceutical composition ofclaim 55, wherein the Wnt surrogate molecule is an agonist of a Wntsignaling pathway.
 63. The method of claim 62, wherein the Wnt surrogatemolecule binds Fzd1, Fzd2, and FZD7, and binds LRP5 and/or LRP6.
 64. Themethod of claim 63, wherein the Wnt surrogate molecule also binds Fzd5and Fzd8.
 65. A method for increasing liver to body weight ratio,promoting liver regeneration, increasing liver cell proliferation ormitosis, decreasing liver fibrosis, optionally following a chronic liverinjury, increasing hepatocyte function, or decreasing coagulation timein liver, comprising providing to a subject an effective amount of thepharmaceutical composition of claim 55, wherein the Wnt surrogatemolecule is an agonist of a Wnt signaling pathway.
 66. The method of anyof claims 57-65, further comprising providing to the subject anantiresorptive agent.
 67. The method of claim 66 for the treatment ofosteoporosis, optionally post-menopausal osteoporosis.
 68. A method forinhibiting or reducing bone resorption in a subject in need thereof,comprising providing to the subject an effective amount of the Wntsurrogate molecule according to any of claims 1-51, the polynucleotideof claim 52, the expression cell of claim 53, the host cell of claim 54,or the pharmaceutical composition of claim 55, wherein the Wnt surrogatemolecule is an agonist of a Wnt signaling pathway.
 69. The method ofclaim 68, further comprising providing to the subject an antiresorptiveagent.
 70. The method of claim 68 or claim 69, wherein the subject hasbeen diagnosed with or is at risk for osteoporosis, optionallypostmenopausal osteoporosis.